Blood and urine multi-omics analysis of the impact of e-vaping, smoking, and cessation: from exposome to molecular responses.

Cigarette smoking is a major preventable cause of morbidity and mortality. While quitting smoking is the best option, switching from cigarettes to non-combustible alternatives (NCAs) such as e-vapor products is a viable harm reduction approach for smokers who would otherwise continue to smoke. A key challenge for the clinical assessment of NCAs is that self-reported product use can be unreliable, compromising the proper evaluation of their risk reduction potential. In this cross-sectional study of 205 healthy volunteers, we combined comprehensive exposure characterization with in-depth multi-omics profiling to compare effects across four study groups: cigarette smokers (CS), e-vapor users (EV), former smokers (FS), and never smokers (NS). Multi-omics analyses included metabolomics, transcriptomics, DNA methylomics, proteomics, and lipidomics. Comparison of the molecular effects between CS and NS recapitulated several previous observations, such as increased inflammatory markers in CS. Generally, FS and EV demonstrated intermediate molecular effects between the NS and CS groups. Stratification of the FS and EV by combustion exposure markers suggested that this position on the spectrum between CS and NS was partially driven by non-compliance/dual use. Overall, this study highlights the importance of in-depth exposure characterization before biological effect characterization for any NCA assessment study.

Suppression of pyrrolidine ring biosynthesis and its effects on gene expression and subsequent accumulation of anatabine in leaves of tobacco (N. tabacum L.).

BACKGROUND: Anatabine, although being one of four major tobacco alkaloids, is never accumulated in high quantity in any of the naturally occurring species from the Nicotiana genus. Previous studies therefore focused on transgenic approaches to synthetize anatabine, most notably by generating transgenic lines with suppressed putrescine methyltransferase (PMT) activity. This led to promising results, but the global gene expression of plants with such distinct metabolism has not been analyzed. In the current study, we describe how these plants respond to topping and the downstream effects on alkaloid biosynthesis. RESULTS: The surge in anatabine accumulation in PMT transgenic lines after topping treatment and its effects on gene expression changes were analyzed. The results revealed increases in expression of isoflavone reductase-like (A622) and berberine bridge-like enzymes (BBLs) oxidoreductase genes, previously shown to be crucial for the final steps of nicotine biosynthesis. We also observed significantly higher methylputrescine oxidase (MPO) expression in all plants subjected to topping treatment. In order to investigate if MPO suppression would have the same effects as that of PMT, we generated transgenic plants. These plants with suppressed MPO expression showed an almost complete drop in leaf nicotine content, whereas leaf anatabine was observed to increase by a factor of ~ 1.6X. CONCLUSION: Our results are the first concrete evidence that suppression of MPO leads to decreased nicotine in favor of anatabine in tobacco roots and that this anatabine is successfully transported to tobacco leaves. Alkaloid transport in plants remains to be investigated to higher detail due to high variation of its efficiency among Nicotiana species and varieties of tobacco. Our research adds important step to better understand pyrrolidine ring biosynthesis and its effects on gene expression and subsequent accumulation of anatabine.

Socio-Ecological Approach to a Forest-Swamp-Savannah Mosaic Landscape Using Remote Sensing and Local Knowledge: a Case Study in the Bas-Ogooué Ramsar Site, Gabon.

Studies of landscape dynamics in protected areas often rely exclusively on remotely-sensed data, leading to bias by neglecting how local inhabitants, who often have a long history of interaction with their environment, perceive and structure the landscape over time. Using a socio-ecological system (SES) approach in a forest-swamp-savannah mosaic within the Bas-Ogooué Ramsar site in Gabon, we assess how human populations participate in landscape dynamics over time. We first conducted a remote sensing analysis to produce a land-cover map representing the biophysical dimension of the SES. This map is based on pixel-oriented classifications, using a 2017 Sentinel-2 satellite image and 610 GPS points, that categorized the landscape in 11 ecological classes. To study the landscape's social dimension, we collected data on local knowledge to understand how local people perceive and use the landscape. These data were collected through 19 semi-structured individual interviews, three focus groups and 3 months of participant observation during an immersive field mission. We developed a systemic approach by combining data on biophysical and social dimensions of the landscape. Our analysis shows that in the absence of continued anthropic interventions, both savannahs and swamps dominated by herbaceous vegetation will experience closure by encroaching woody vegetation, leading to eventual biodiversity loss. Our methodology based on an SES approach to landscapes could improve the conservation programs developed by Ramsar site managers. Designing actions at the local scale, rather than applying one set of actions to the entire protected area, allows the integration of human perceptions, practices and expectations, a challenge that is more than essential in the context of global change.

Fractionation and Extraction Optimization of Potentially Valuable Compounds and Their Profiling in Six Varieties of Two Nicotiana Species.

There is an increasingly urgent call to shift industrial processes from fossil fuel feedstock to sustainable bio-based resources. This change becomes of high importance considering new budget requirements for a carbon-neutral economy. Such a transformation can be driven by traditionally used plants that are able to produce large amounts of valuable biologically relevant secondary metabolites. Tobacco plants can play a leading role in providing value-added products in remote areas of the world. In this study, we propose a non-exhaustive list of compounds with potential economic interest that can be sourced from the tobacco plant. In order to optimize extraction methodologies, we first analyzed their physico-chemical properties using rapid solubility tests and high-resolution microfractionation techniques. Next, to identify an optimal extraction for a selected list of compounds, we compared 13 different extraction method-solvent combinations. We proceeded with profiling some of these compounds in a total of six varieties from Nicotiana tabacum and Nicotiana rustica species, identifying the optimal variety for each. The estimated expected yields for each of these compounds demonstrate that tobacco plants can be a superior source of valuable compounds with diverse applications beyond nicotine. Among the most interesting results, we found high variability of anatabine content between species and varieties, ranging from 287 to 1699 µg/g. In addition, we found that CGA (1305 µg/g) and rutin (7910 µg/g) content are orders of magnitude lower in the Burley variety as compared to all others.

Application of Secondary Electrospray Ionization Coupled with High-Resolution Mass Spectrometry in Chemical Characterization of Thermally Generated Aerosols.

Inhalation as a route for administering drugs and dietary supplements has garnered significant attention over the past decade. We performed real-time analyses of aerosols using secondary electrospray ionization (SESI) technology interfaced with high-resolution mass spectrometry (HRMS), primarily developed for exhaled breath analysis with the goal to detect the main aerosol constituents. Several commercially available inhalation devices containing caffeine, melatonin, cannabidiol, and vitamin B12 were tested. Chemical characterization of the aerosols produced by these devices enabled detection of the main constituents and screening for potential contaminants, byproducts, and impurities in the aerosol. In addition, a programmable syringe pump was connected to the SESI-HRMS system to monitor aerosolized active pharmaceutical ingredients (APIs) such as chloroquine, hydroxychloroquine, and azithromycin. This setup allowed us to detect caffeine, melatonin, hydroxychloroquine, chloroquine, and cannabidiol in the produced aerosols. Azithromycin and vitamin B12 in the aerosols could not be detected; however, our instrument setup enabled the detection of vitamin B12 breakdown products that were generated during the aerosolization process. Positive control was realized by liquid chromatography-HRMS analyses. The compounds detected in the aerosol were confirmed by exact mass measurements of the protonated and/or deprotonated species, as well as their respective collision-induced dissociation tandem mass spectra. These results reveal the potential wide application of this technology for the real-time monitoring of aerosolized active pharmaceutical ingredients that can be administered through the inhalation route.

Pulmonary Delivery of Aerosolized Chloroquine and Hydroxychloroquine to Treat COVID-19: In Vitro Experimentation to Human Dosing Predictions.

In vitro screening for pharmacological activity of existing drugs showed chloroquine and hydroxychloroquine to be effective against severe acute respiratory syndrome coronavirus 2. Oral administration of these compounds to obtain desired pulmonary exposures resulted in dose-limiting systemic toxicity in humans. However, pulmonary drug delivery enables direct and rapid administration to obtain higher local tissue concentrations in target tissue. In this work, inhalable formulations for thermal aerosolization of chloroquine and hydroxychloroquine were developed, and their physicochemical properties were characterized. Thermal aerosolization of 40 mg/mL chloroquine and 100 mg/mL hydroxychloroquine formulations delivered respirable aerosol particle sizes with 0.15 and 0.33 mg per 55 mL puff, respectively. In vitro toxicity was evaluated by exposing primary human bronchial epithelial cells to aerosol generated from Vitrocell. An in vitro exposure to 7.24 µg of chloroquine or 7.99 µg hydroxychloroquine showed no significant changes in cilia beating, transepithelial electrical resistance, and cell viability. The pharmacokinetics of inhaled aerosols was predicted by developing a physiologically based pharmacokinetic model that included a detailed species-specific respiratory tract physiology and lysosomal trapping. Based on the model predictions, inhaling emitted doses comprising 1.5 mg of chloroquine or 3.3 mg hydroxychloroquine three times a day may yield therapeutically effective concentrations in the lung. Inhalation of higher doses further increased effective concentrations in the lung while maintaining lower systemic concentrations. Given the theoretically favorable risk/benefit ratio, the clinical significance for pulmonary delivery of aerosolized chloroquine and hydroxychloroquine to treat COVID-19 needs to be established in rigorous safety and efficacy studies. Graphical abstract.

Assessment of in vitro kinetics and biological impact of nebulized trehalose on human bronchial epithelium.

Trehalose is added in drug formulations to act as fillers or improve aerosolization performance. Its characteristics as a carrier molecule have been explored; however, the fate of trehalose in human airway tissues has not been thoroughly investigated. Here, we investigated the fate of nebulized trehalose using in vitro human air-liquid bronchial epithelial cultures. First, a tracing experiment was conducted using 13C12-trehalose; we measured trehalose distribution in different culture compartments (apical surface liquid, epithelial culture, and basal side medium) at various time points following acute exposure to 13C12-labeled trehalose. We found that 13C12-trehalose was metabolized into 13C6-glucose. The data was then used to model the kinetics of trehalose disappearance from the apical surface of bronchial cultures. Secondly, we evaluated the potential adverse effects of nebulized trehalose on the bronchial cultures after they were acutely exposed to nebulized trehalose up to a level just below its solubility limit (50 g/100 g water). We assessed the ciliary beating frequency and histological characteristics. We found that nebulized trehalose did not lead to marked alteration in ciliary beating frequency and morphology of the epithelial cultures. The in vitro testing approach used here may enable the early selection of excipients for future development of inhalation products.

A Literature Review and Framework Proposal for Halitosis Assessment in Cigarette Smokers and Alternative Nicotine-Delivery Products Users.

Halitosis is a health condition which counts cigarette smoking (CS) among its major risk factors. Cigarette smoke can cause an imbalance in the oral bacterial community, leading to several oral diseases and conditions, including intraoral halitosis. Although the best approach to decrease smoking-related health risks is quitting smoking, this is not feasible for many smokers. Switching to potentially reduced-risk products, like electronic vapor products (EVP) or heated tobacco products (HTP), may help improve the conditions associated with CS. To date, there have been few systematic studies on the effects of CS on halitosis and none have assessed the effects of EVP and HTP use. Self-assessment studies have shown large limitations owing to the lack of reliability in the participants' judgment. This has compelled the scientific community to develop a strategy for meaningful assessment of these new products in comparison with cigarettes. Here, we compiled a review of the existing literature on CS and halitosis and propose a 3-layer approach that combines the use of the most advanced breath analysis techniques and multi-omics analysis to define the interactions between oral bacterial species and their role in halitosis both in vitro and in vivo. Such an approach will allow us to compare the effects of different nicotine-delivery products on oral bacteria and quantify their impact on halitosis. Defining the impact of alternative nicotine-delivery products on intraoral halitosis and its associated bacteria will help the scientific community advance a step further toward understanding the safety of these products and their potentiall risks for consumers.

Analysis of chemical deposits on tooth enamel exposed to total particulate matter from cigarette smoke and tobacco heating system 2.2 aerosol by novel GC-MS deconvolution procedures.

Tobacco smoking contributes to tooth discoloration. Pigmented compounds in the smoke generated by combustion of tobacco can cause discoloration of dental hard tissues. However, aerosols from heated tobacco products cause less discoloration than cigarette smoke (CS) in vitro. The objective of the present study was to optimize a method for extracting the colored chemical compounds deposited on tooth enamel following exposure to total particulate matter (TPM) from CS or a heated tobacco product (Tobacco Heating System [THS] 2.2), analyze the extracts by gas chromatography coupled to time-of-flight mass spectrometry, and identify the key chemicals associated with tooth discoloration. Sixty bovine enamel blocks were exposed for 2 weeks to TPM from CS or THS 2.2 aerosol or to artificial saliva as a control. Brushing without toothpaste and color measurements were performed each week. Noticeable discoloration of enamel was observed following exposure to CS TPM. The discoloration following exposure to THS 2.2 aerosol TPM or artificial saliva was not distinguishable to the eye (ΔE < 3.3). Carbon disulfide was used to extract surface-deposited chemicals. Untargeted analyses were followed by partial least squares correlation against discoloration scores (R2 = 0.96). Eleven compounds had variable importance in projection scores greater than 2. Discriminant autocorrelation matrix calculation of their mass spectral information identified eight of the eleven compounds as terpenoids. None of the compounds were related to nicotine. Several of these compounds were also detected in THS 2.2 aerosol TPM-exposed enamel, but at lower levels, in line with our findings showing less discoloration. Compared with CS TPM exposure, THS 2.2 aerosol TPM exposure resulted in lower deposition of color-related compounds on enamel surface, consistent with minimal discoloration of dental enamel.

Delivery efficiencies of constituents of combustion-derived aerosols across the air-liquid interface during in vitro exposures.

In vitro aerosol exposure of epithelial cells grown at the air-liquid interface is an experimental methodology widely used in respiratory toxicology. The exposure depends to a large part on the physicochemical properties of individual aerosol constituents, as they determine the transfer kinetics from the aerosol into the cells. We characterized the transfer of 70 cigarette smoke constituents from the smoke into aqueous samples exposed in the Vitrocell® 24/48 aerosol exposure system. The amounts of these compounds in the applied smoke were determined by trapping whole smoke in N,N-dimethylformamide and then compared with their amounts in smoke-exposed, phosphate-buffered saline, yielding compound specific delivery efficiencies. Delivery efficiencies of different smoke constituents differed by up to five orders of magnitude, which indicates that the composition of the applied smoke is not necessarily representative for the delivered smoke. Therefore, dose metrics for in vitro exposure experiments should, if possible, be based on delivered and not applied doses. A comparison to literature on in vivo smoke retention in the respiratory tract indicated that the same applies for smoke retention in the respiratory tract.

Prediction Models of Retention Indices for Increased Confidence in Structural Elucidation during Complex Matrix Analysis: Application to Gas Chromatography Coupled with High-Resolution Mass Spectrometry.

Monitoring of volatile and semivolatile compounds was performed using gas chromatography (GC) coupled to high-resolution electron ionization mass spectrometry, using both headspace and liquid injection modes. A total of 560 reference compounds, including 8 odd n-alkanes, were analyzed and experimental linear retention indices (LRI) were determined. These reference compounds were randomly split into training (n = 401) and test (n = 151) sets. LRI for all 552 reference compounds were also calculated based upon computational Quantitative Structure-Property Relationship (QSPR) models, using two independent approaches RapidMiner (coupled to Dragon) and ACD/ChromGenius software. Correlation coefficients for experimental versus predicted LRI values calculated for both training and test set compounds were calculated at 0.966 and 0.949 for RapidMiner and at 0.977 and 0.976 for ACD/ChromGenius, respectively. In addition, the cross-validation correlation was calculated at 0.96 from RapidMiner and the residual standard error value obtained from ACD/ChromGenius was 53.635. These models were then used to predict LRI values for several thousand compounds reported present in tobacco and tobacco-related fractions, plus a range of specific flavor compounds. It was demonstrated that using the mean of the LRI values predicted by RapidMiner and ACD/ChromGenius, in combination with accurate mass data, could enhance the confidence level for compound identification from the analysis of complex matrixes, particularly when the two predicted LRI values for a compound were in close agreement. Application of this LRI modeling approach to matrixes with unknown composition has already enabled the confirmation of 23 postulated compounds, demonstrating its ability to facilitate compound identification in an analytical workflow. The goal is to reduce the list of putative candidates to a reasonable relevant number that can be obtained and measured for confirmation.

Characterization of the Vitrocell® 24/48 in vitro aerosol exposure system using mainstream cigarette smoke.

BACKGROUND: Only a few exposure systems are presently available that enable cigarette smoke exposure of living cells at the air-liquid interface, of which one of the most versatile is the Vitrocell® system (Vitrocell® Systems GmbH). To assess its performance and optimize the exposure conditions, we characterized a Vitrocell® 24/48 system connected to a 30-port carousel smoking machine. The Vitrocell® 24/48 system allows for simultaneous exposure of 48 cell culture inserts using dilution airflow rates of 0-3.0 L/min and exposes six inserts per dilution. These flow rates represent cigarette smoke concentrations of 7-100%. RESULTS: By characterizing the exposure inside the Vitrocell® 24/48, we verified that (I) the cigarette smoke aerosol distribution is uniform across all inserts, (II) the utility of Vitrocell® crystal quartz microbalances for determining the online deposition of particle mass on the inserts, and (III) the amount of particles deposited per surface area and the amounts of trapped carbonyls and nicotine were concentration dependent. At a fixed dilution airflow of 0.5 L/min, the results showed a coefficient of variation of 12.2% between inserts of the Vitrocell® 24/48 module, excluding variations caused by different runs. Although nicotine and carbonyl concentrations were linear over the tested dilution range, particle mass deposition increased nonlinearly. The observed effect on cell viability was well-correlated with increasing concentration of cigarette smoke. CONCLUSIONS: Overall, the obtained results highlight the suitability of the Vitrocell® 24/48 system to assess the effect of cigarette smoke on cells under air-liquid interface exposure conditions, which is closely related to the conditions occurring in human airways.

Measurement of ultrasonic scattering attenuation in austenitic stainless steel welds: realistic input data for NDT numerical modeling.

Multipass welds made of 316L stainless steel are specific welds of the primary circuit of pressurized water reactors in nuclear power plants. Because of their strong heterogeneous and anisotropic nature due to grain growth during solidification, ultrasonic waves may be greatly deviated, split and attenuated. Thus, ultrasonic assessment of the structural integrity of such welds is quite complicated. Numerical codes exist that simulate ultrasonic propagation through such structures, but they require precise and realistic input data, as attenuation coefficients. This paper presents rigorous measurements of attenuation in austenitic weld as a function of grain orientation. In fact attenuation is here mainly caused by grain scattering. Measurements are based on the decomposition of experimental beams into plane-wave angular spectra and on the modeling of the ultrasonic propagation through the material. For this, the transmission coefficients are calculated for any incident plane wave on an anisotropic plate. Two different hypotheses on the welded material are tested: first it is considered as monoclinic, and then as triclinic. Results are analyzed, and validated through comparison to theoretical predictions of related literature. They underline the great importance of well-describing the anisotropic structure of austenitic welds for UT modeling issues.

Topographical body fat distribution links to amino acid and lipid metabolism in healthy obese women [corrected].

Visceral adiposity is increasingly recognized as a key condition for the development of obesity related disorders, with the ratio between visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) reported as the best correlate of cardiometabolic risk. In this study, using a cohort of 40 obese females (age: 25-45 y, BMI: 28-40 kg/m(2)) under healthy clinical conditions and monitored over a 2 weeks period we examined the relationships between different body composition parameters, estimates of visceral adiposity and blood/urine metabolic profiles. Metabonomics and lipidomics analysis of blood plasma and urine were employed in combination with in vivo quantitation of body composition and abdominal fat distribution using iDXA and computerized tomography. Of the various visceral fat estimates, VAT/SAT and VAT/total abdominal fat ratios exhibited significant associations with regio-specific body lean and fat composition. The integration of these visceral fat estimates with metabolic profiles of blood and urine described a distinct amino acid, diacyl and ether phospholipid phenotype in women with higher visceral fat. Metabolites important in predicting visceral fat adiposity as assessed by Random forest analysis highlighted 7 most robust markers, including tyrosine, glutamine, PC-O 44∶6, PC-O 44∶4, PC-O 42∶4, PC-O 40∶4, and PC-O 40∶3 lipid species. Unexpectedly, the visceral fat associated inflammatory profiles were shown to be highly influenced by inter-days and between-subject variations. Nevertheless, the visceral fat associated amino acid and lipid signature is proposed to be further validated for future patient stratification and cardiometabolic health diagnostics.

Dose-response plasma appearance of green tea catechins in adults.

SCOPE: Tea is an infusion of the Camellia sinensis leaves. The most prevalent bioactive compounds in green tea are catechins (C), which are of great interest for their potential health-promoting effects. However, metabolism and bioavailability of C are not fully understood. METHODS AND RESULTS: This study investigates the human bioavailability (plasma appearance) of C after drinking three doses of infused green tea in a randomized cross-over design. The sum of area under the curve increased between the small (0.75% w/v, 180 mg total C) and medium (1.25%) dose of ingested green tea but not between the medium and the high (1.75%) dose. The overall pattern for the sum of C did not reflect the fate of individual C. While (-)-epigallocatechin and 4'-O-Me-epigallocatechin showed saturation in plasma between the medium and high green tea doses, (-)-epigallocatechin gallate and (-)-epicatechin did not "saturate" and increased proportionally with the ingested dose. Regardless of the dose, C appeared rapidly in plasma as monophasic curves, suggesting absorption in the small intestine and minimal entero-hepatic circulation. CONCLUSION: As a conclusion, when studying dose response of polyphenols and metabolites, one must look not only at the overall pattern of plasma appearance, but also at data specific for each metabolite.

A whole-grain-rich diet reduces urinary excretion of markers of protein catabolism and gut microbiota metabolism in healthy men after one week.

Epidemiological studies consistently find that diets rich in whole-grain (WG) cereals lead to decreased risk of disease compared with refined grain (RG)-based diets. Aside from a greater amount of fiber and micronutrients, possible mechanisms for why WGs may be beneficial for health remain speculative. In an exploratory, randomized, researcher-blinded, crossover trial, we measured metabolic profile differences between healthy participants eating a diet based on WGs compared with a diet based on RGs. Seventeen healthy adult participants (11 female, 6 male) consumed a controlled diet based on either WG-rich or RG-rich foods for 2 wk, followed by the other diet after a 5-wk washout period. Both diets were the same except for the use of WG (150 g/d) or RG foods. The metabolic profiles of plasma, urine, and fecal water were measured using (1)H-nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry (plasma only). After 1 wk of intervention, the WG diet led to decreases in urinary excretion of metabolites related to protein catabolism (urea, methylguanadine), lipid (carnitine and acylcarnitines) and gut microbial (4-hydroxyphenylacetate, trimethylacetate, dimethylacetate) metabolism in men compared with the same time point during the RG intervention. There were no differences between the interventions after 2 wk. Urinary urea, carnitine, and acylcarnitine were lower at wk 1 of the WG intervention relative to the RG intervention in all participants. Fecal water short-chain fatty acids acetate and butyrate were relatively greater after the WG diet compared to the RG diet. Although based on a small population and for a short time period, these observations suggest that a WG diet may affect protein metabolism.

Specific dietary preferences are linked to differing gut microbial metabolic activity in response to dark chocolate intake.

Systems biology approaches are providing novel insights into the role of nutrition for the management of health and disease. In the present study, we investigated if dietary preference for dark chocolate in healthy subjects may lead to different metabolic response to daily chocolate consumption. Using NMR- and MS-based metabolic profiling of blood plasma and urine, we monitored the metabolic response of 10 participants stratified as chocolate desiring and eating regularly dark chocolate (CD) and 10 participants stratified as chocolate indifferent and eating rarely dark chocolate (CI) to a daily consumption of 50 g of dark chocolate as part of a standardized diet over a one week period. We demonstrated that preference for chocolate leads to different metabolic response to chocolate consumption. Daily intake of dark chocolate significantly increased HDL cholesterol by 6% and decreased polyunsaturated acyl ether phospholipids. Dark chocolate intake could also induce an improvement in the metabolism of long chain fatty acid, as noted by a compositional change in plasma fatty acyl carnitines. Moreover, a relationship between regular long-term dietary exposure to a small amount of dark chocolate, gut microbiota, and phenolics was highlighted, providing novel insights into biological processes associated with cocoa bioactives.

A protein-leucine supplement increases branched-chain amino acid and nitrogen turnover but not performance.

PURPOSE: This study aimed to determine the effect of postexercise protein-leucine coingestion with CHO-lipid on subsequent high-intensity endurance performance and to investigate candidate mechanisms using stable isotope methods and metabolomics. METHODS: In this double-blind, randomized, crossover study, 12 male cyclists ingested a leucine/protein/CHO/fat supplement (LEUPRO 7.5/20/89/22 g · h(-1), respectively) or isocaloric CHO/fat control (119/22 g · h(-1)) 1-3 h after exercise during a 6-d training block (intense intervals, recovery, repeated-sprint performance rides). Daily protein intake was clamped at 1.9 g · kg(-1) · d(-1) (LEUPRO) and 1.5 g · kg(-1) · d(-1) (control). Stable isotope infusions (1-(13)C-leucine and 6,6-(2)H2-glucose), mass spectrometry-based metabolomics, and nitrogen balance methods were used to determine the effects of LEUPRO on whole-body branched-chain amino acid (BCAA) and glucose metabolism and protein turnover. RESULTS: After exercise, LEUPRO increased BCAA levels in plasma (2.6-fold; 90% confidence limits = ×/÷ 1.1) and urine (2.8-fold; ×/÷ 1.2) and increased products of BCAA metabolism plasma acylcarnitine C5 (3.0-fold; ×/÷ 0.9) and urinary leucine (3.6-fold; ×/÷ 1.3) and ß-aminoisobutyrate (3.4-fold; ×/÷ 1.4), indicating that ingesting ~10 g leucine per hour during recovery exceeds the capacity to metabolize BCAA. Furthermore, LEUPRO increased leucine oxidation (5.6-fold; ×/÷ 1.1) and nonoxidative disposal (4.8-fold; ×/÷ 1.1) and left leucine balance positive relative to control. With the exception of day 1 (LEUPRO = 17 ± 20 mg N · kg(-1), control = -90 ± 44 mg N · kg(-1)), subsequent (days 2-5) nitrogen balance was positive for both conditions (LEUPRO = 130 ± 110 mg N · kg(-1), control = 111 ± 86 mg N · kg(-1)). Compared with control feeding, LEUPRO lowered the serum creatine kinase concentration by 21%-25% (90% confidence limits = ± 14%), but the effect on sprint power was trivial (day 4 = 0.4% ± 1.0%, day 6 = -0.3% ± 1.0%). CONCLUSIONS: Postexercise protein-leucine supplementation saturates BCAA metabolism and attenuates tissue damage, but effects on subsequent intense endurance performance may be inconsequential under conditions of positive daily nitrogen balance.

Metabolic phenotyping of the Crohn's disease-like IBD etiopathology in the TNF(ΔARE/WT) mouse model.

The underlying biochemical consequences of inflammatory bowel disease (IBD) on the systemic and gastrointestinal metabolism have not yet been fully elucidated but could help to better understand the disease pathogenesis and to identify tissue-specific markers associated with the different disease stages. Here, we applied a metabonomic approach to monitor metabolic events associated with the gradual development of Crohn's disease (CD)-like ileitis in the TNF(ΔARE/WT) mouse model. Metabolic profiles of different intestinal compartments from the age of 4 up to 24 weeks were generated by combining proton nuclear magnetic resonance ((1)H NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS). From 8 weeks onward, mice developed CD similar to the immune and tissue-related phenotype of human CD with ileal involvement, including ileal histological abnormalities, reduced fat mass and body weight, as well as hallmarks of malabsorption with higher energy wasting. The metabonomic approach highlighted shifts in the intestinal lipid metabolism concomitant to the histological onset of inflammation. Moreover, the advanced disease status was characterized by a significantly altered metabolism of cholesterol, triglycerides, phospholipids, plasmalogens, and sphingomyelins in the inflamed tissue (ileum) and the adjacent intestinal parts (proximal colon). These results describe different biological processes associated with the disease onset, including modifications of the general cell membrane composition, alteration of energy homeostasis, and finally the generation of inflammatory lipid mediators. Taken together, this provides novel insights into IBD-related alterations of specific lipid-dependant processes during inflammatory states.

Identification of novel circulating coffee metabolites in human plasma by liquid chromatography-mass spectrometry.

This study reports a liquid chromatography-mass spectrometry method for the detection of polyphenol-derived metabolites in human plasma without enzymatic treatment after coffee consumption. Separation of available standards was achieved by reversed-phase ultra performance liquid chromatography and detection was performed by high resolution mass spectrometry in negative electrospray ionization mode. This analytical method was then applied for the identification and relative quantification of circulating coffee metabolites. A total of 34 coffee metabolites (mainly reduced, sulfated and methylated forms of caffeic acid, coumaric acid, caffeoylquinic acid and caffeoylquinic acid lactone) were identified based on mass accuracy (<4 ppm for most metabolites), specific fragmentation pattern and co-chromatography (when standard available). Among them, 19 circulating coffee metabolites were identified for the first time in human plasma such as feruloylquinic acid lactone, sulfated and glucuronidated forms of feruloylquinic acid lactone and sulfated forms of coumaric acid. Phenolic acid derivatives such as dihydroferulic acid, dihydroferulic acid 4'-O-sulfate, caffeic acid 3'-O-sulfate, dimethoxycinnamic acid, dihydrocaffeic acid and coumaric acid O-sulfate appeared to be the main metabolites circulating in human plasma after coffee consumption. The described method is a sensitive and reliable approach for the identification of coffee metabolites in biological fluids. In future, this analytical method will give more confidence in compound identification to provide a more comprehensive assessment of coffee polyphenol bioavailability studies in humans.