Breath gas monitoring during a glucose challenge by a combined PTR-QMS/GC×GC-TOFMS approach for the verification of potential volatile biomarkers.

Breath gas profiles, which reflect metabolic disorders like diabetes, are the subject of scientific focus. Nevertheless, profiling is still a challenging task that requires complex and standardized methods. This study was carried out to verify breath gas patterns that were obtained in previous proton-transfer reaction-quadrupole mass spectrometry (PTR-QMS) studies and that can be linked to glucose metabolism. An experimental setup using simultaneous PTR-QMS and complementary highly time-resolved needle trap micro extraction (NTME) combined with comprehensive 2D gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS) was established for the analysis of highly polar volatile organic compounds (VOCs). The method was applied to the breath gas analysis of three volunteers during a glucose challenge, whereby subjects ingested a glucose solution orally. Challenge responsive PTR-QMS target VOCs could be linked to small n-carbonic (C2-C4) alcohols and short chain fatty acids (SCFA). Specific isomers could be identified by simultaneously applied NTME-GC×GC-TOFMS and further verified by their characteristic time profiles and concentrations. The identified VOCs potentially originate from bacteria that are found in the oral cavity and gastrointestinal tract. In this study breath gas monitoring enabled the identification of potential VOC metabolites that can be linked to glucose metabolism.

Diet-induced and mono-genetic obesity alter volatile organic compound signature in mice.

The prevalence of obesity is still rising in many countries, resulting in an increased risk of associated metabolic diseases. In this study we aimed to describe the volatile organic compound (VOC) patterns symptomatic for obesity. We analyzed high fat diet (HFD) induced obese and mono-genetic obese mice (global knock-in mutation in melanocortin-4 receptor MC4R-ki). The source strengths of 208 VOCs were analyzed in ad libitum fed mice and after overnight food restriction. Volatiles relevant for a random forest-based separation of obese mice were detected (26 in MC4R-ki, 22 in HFD mice). Eight volatiles were found to be important in both obesity models. Interestingly, by creating a partial correlation network of the volatile metabolites, the chemical and metabolic origins of several volatiles were identified. HFD-induced obese mice showed an elevation in the ketone body acetone and acrolein, a marker of lipid peroxidation, and several unidentified volatiles. In MC4R-ki mice, several yet-unidentified VOCs were found to be altered. Remarkably, the pheromone (methylthio)methanethiol was found to be reduced, linking metabolic dysfunction and reproduction. The signature of volatile metabolites can be instrumental in identifying and monitoring metabolic disease states, as shown in the screening of the two obese mouse models in this study. Our findings show the potential of breath gas analysis to non-invasively assess metabolic alterations for personalized diagnosis.

Online breath gas analysis in unrestrained mice by hs-PTR-MS.

The phenotyping of genetic mouse models for human disorders may greatly benefit from breath gas analysis as a noninvasive tool to identify metabolic alterations in mice. Phenotyping screens such as the German Mouse Clinic demand investigations in unrestrained mice. Therefore, we adapted a breath screen in which exhaled volatile organic compounds (VOCs) were online monitored by proton transfer reaction mass spectrometry (hs-PTR-MS). The source strength of VOCs was derived from the dynamics in the accumulation profile of exhaled VOCs of a single mouse in a respirometry chamber. A careful survey of the accumulation revealed alterations in the source strength due to confounders, e.g., urine and feces. Moreover changes in the source strength of humidity were triggered by changes in locomotor behavior as mice showed a typical behavioral pattern from activity to settling down in the course of subsequent accumulation profiles. We demonstrated that metabolic changes caused by a dietary intervention, e.g., after feeding a high-fat diet (HFD) a sample of 14 male mice, still resulted in a statistically significant shift in the source strength of exhaled VOCs. Applying a normalization which was derived from the distribution of the source strength of humidity and accounted for varying locomotor behaviors improved the shift. Hence, breath gas analysis may provide a noninvasive, fast access to monitor the metabolic adaptation of a mouse to alterations in energy balance due to overfeeding or fasting and dietary macronutrient composition as well as a high potential for systemic phenotyping of mouse mutants, intervention studies, and drug testing in mice.

Innovations in phenotyping of mouse models in the German Mouse Clinic.

Under the label of the German Mouse Clinic (GMC), a concept has been developed and implemented that allows the better understanding of human diseases on the pathophysiological and molecular level. This includes better understanding of the crosstalk between different organs, pleiotropy of genes, and the systemic impact of envirotypes and drugs. In the GMC, experts from various fields of mouse genetics and physiology, in close collaboration with clinicians, work side by side under one roof. The GMC is an open-access platform for the scientific community by providing phenotypic analysis in bilateral collaborations ("bottom-up projects") and as a partner and driver in international large-scale biology projects ("top-down projects"). Furthermore, technology development is a major topic in the GMC. Innovative techniques for primary and secondary screens are developed and implemented into the phenotyping pipelines (e.g., detection of volatile organic compounds, VOCs).

Human breath gas analysis in the screening of gestational diabetes mellitus.

BACKGROUND: We present a pilot study on the feasibility of the application and advantages of online, noninvasive breath gas analysis (BGA) by proton transfer reaction quadrupole mass spectrometry for the screening of gestational diabetes mellitus (GDM) in 52 pregnant women by means of an oral glucose tolerance test (OGTT). SUBJECTS AND METHODS: We collected and identified samples of end-tidal breath gas from patients during OGTT. Time evolution parameters of challenge-responsive volatile organic compounds (VOCs) in human breath gas were estimated. Multivariate analysis of variance and permutation analysis were used to assess feasibility of BGA as a diagnostic tool for GDM. RESULTS: Standard OGTT diagnosis identified pregnant women as having GDM (n = 8), impaired glucose tolerance (n = 12), and normal glucose tolerance (n = 32); a part of this latter group was further subdivided into a "marginal" group (n = 9) because of a marginal high 1-h or 2-h OGTT value. We observed that OGTT diagnosis (four metabolic groups) could be mapped into breath gas data. The time evolution of oxidation products of glucose and lipids, acetone metabolites, and thiols in breath gas after a glucose challenge was correlated with GDM diagnosis (P = 0.035). Furthermore, basal (fasting) values of dimethyl sulfide and values of methanol in breath gas were inversely correlated with phenotype characteristics such as homeostasis model assessment of insulin resistance index (R = -0.538; P = 0.0002, P(corrected) = 0.0034) and pregestational body mass index (R = -0.433; P = 0.0013, P(corrected) = 0.022). CONCLUSIONS: Noninvasive BGA in challenge response studies was successfully applied to GDM diagnosis and offered an insight into metabolic pathways involved. We propose a new approach to the identification of diagnosis thresholds for GDM screening.

The dynamic range of the human metabolome revealed by challenges.

Metabolic challenge protocols, such as the oral glucose tolerance test, can uncover early alterations in metabolism preceding chronic diseases. Nevertheless, most metabolomics data accessible today reflect the fasting state. To analyze the dynamics of the human metabolome in response to environmental stimuli, we submitted 15 young healthy male volunteers to a highly controlled 4 d challenge protocol, including 36 h fasting, oral glucose and lipid tests, liquid test meals, physical exercise, and cold stress. Blood, urine, exhaled air, and breath condensate samples were analyzed on up to 56 time points by MS- and NMR-based methods, yielding 275 metabolic traits with a focus on lipids and amino acids. Here, we show that physiological challenges increased interindividual variation even in phenotypically similar volunteers, revealing metabotypes not observable in baseline metabolite profiles; volunteer-specific metabolite concentrations were consistently reflected in various biofluids; and readouts from a systematic model of ß-oxidation (e.g., acetylcarnitine/palmitylcarnitine ratio) showed significant and stronger associations with physiological parameters (e.g., fat mass) than absolute metabolite concentrations, indicating that systematic models may aid in understanding individual challenge responses. Due to the multitude of analytical methods, challenges and sample types, our freely available metabolomics data set provides a unique reference for future metabolomics studies and for verification of systems biology models.

Radiation dose assessment of exposure to depleted uranium.

Depleted uranium (DU) is claimed to contribute to human health problems, known as the Gulf War Syndrome and the Balkan Syndrome. Quantitative radiation dose is required to estimate the health risk of DU materials. The influences of the solubility parameters in the human alimentary tract and the respiratory tract systems and the aerosol particles size on the radiation dose of DU materials were evaluated. The dose conversion factor of daily urinary excretion of DU is provided. The retention and excretion of DU in the human body after a contamination at a wound site were predicted. Dose coefficients of DU after ingestion and inhalation were calculated using the solubility parameters of the DU corrosion products in simulated gastric and simulated lung fluid, which were determined in the Helmholtz Zentrum München. (238)U is the main radiation dose contributor per 1 Bq of DU materials. The dose coefficients of DU materials were estimated to be 3.5 x 10(-8) and 2.1 x 10(-6) Sv Bq(-1) after ingestion and inhalation for members of the public. The ingestion dose coefficient of DU materials is about 75% of the natural uranium value. The inhalation dose coefficient of DU material is in between those for Type M and Type S according to the category for inhaled materials defined by the International Commission on Radiological Protection. Radiation dose possibly received from DU materials can directly be estimated by using the dose conversion factor provided in this study, if daily urinary excretion of DU is measured.

Investigations on the solubility of corrosion products on depleted uranium projectiles by simulated body fluids and the consequences on dose assessment.

Ingestion and inhalation of corrosion products covering weathered penetrators made of depleted uranium (DU) represent potential radiological exposure pathways. In order to study the bioavailability of these corrosion products, their solubility was determined using simulated gastric and pulmonary juices. About 75 and 36% of the uranium in the corrosion products were found to be soluble in simulated gastric and pulmonary juices, respectively. The effective dose coefficient for adults after ingestion was calculated to be 0.61 muSv mg(-1) DU. This compares to an effective dose coefficient for an adult of 0.71 muSv mg(-1) for DU materials given by the World Health Organization (WHO). The effective dose coefficient for inhalation was calculated to be 3.7 x 10(-6 )Sv Bq(-1) for workers and 5.3 x 10(-6 )Sv Bq(-1) for members of the public, respectively, which is between those of particles of Types M and S as defined by the International Commission on Radiological Protection (ICRP). The speciation of the corrosion products was investigated by time-of-flight secondary ion mass spectrometry (TOF-SIMS). The mean oxidation state of uranium was found to be 4.6, which suggests that the uranium in the corrosion products consists of a mixture of U(IV) and U(VI) species.

Orientation modulation of a synthetic polypeptide in self-assembled monolayers: a TOF-SIMS study.

Structure and orientation of molecules are key properties of functionalized surfaces. Using time-of-flight secondary ion mass spectrometry (TOF-SIMS), here we investigate how to modulate these parameters upon the immobilization process varying the conditions of self-assembly. The molecule of interest, a template-assembled synthetic protein (TASP), consists of a central peptide ring with orthogonally arranged residues. Thioalkane chains allow the directed self-assembly of the molecule on a gold surface; four serine residues on the opposite side of the ring can be used as anchoring sites for various functional sensing molecules. The TASP conformation and its orientation in self-assembled monolayers (SAMs) play a central role for the accessibility of these serine residues. To study the influence of the self-assembly conditions, two series of samples were prepared. Pure TASP monolayers of different surface densities are compared to mixed TASP/alkanethiol monolayers prepared by sequential adsorption varying sequence and particular incubation times as well as by coadsorption modifying incubation times and TASP/alkanethiol mass ratios. Switching the TASP orientation from a state where the molecules are lying flat on the surface to an upright orientation turned out to be possible by inserting the TASP into a preformed alkanethiol monolayer of an appropriate surface density. This study demonstrates that TOF-SIMS is an excellent tool not only to investigate the surface composition, but also the molecular structure of functionalized surfaces.

Effect of water treatment on analyte and matrix ion yields in matrix-assisted time-of-flight secondary ion mass spectrometry: the case of insulin in and on hydroxycinnamic acid.

A systematic study was performed to identify the origin of surprisingly high analyte-to-matrix yield ratios recently observed in time-of-flight secondary ion mass spectrometry (TOF-SIMS) analysis of oligo- and polypeptides mixed in matrices of alpha-cyano-4-hydroxycinnamic acid (4HCCA). Several sets of samples of porcine insulin in 4HCCA (1:3100 molar) were prepared from liquid solutions by a nebuliser technique, with more than one order of magnitude variation in sprayed material (substrate silicon). Following different periods of storage in air and/or vacuum as well as exposure to high-purity water, TOF-SIMS analysis was performed under oblique impact of 22 keV SF5+. Treatment with water involved either deposition of a droplet covering the whole sample for times between 1 and 20 min or spraying with water in droplet equivalent quantities. The analyte and matrix molecules were detected as protonated molecules (insulin also in doubly protonated form). Even the as-prepared samples usually showed insulin-to-4HCCA yield ratios exceeding the molar ratio of the mixed material. Upon ageing in vacuum the matrix ion yields remained constant but the analyte yields decreased, partly due to break-up of intrachain disulfide bonds. Water treatment resulted in a pronounced decrease in the 4HCCA yield, typically by a factor of five, in parallel with an increase of the insulin yield, by up to a factor of four. Evidence is provided that these changes occur concurrently with a partial dissolution of 4HCCA at the sample surface. The enhanced insulin yield was not correlated with the Na+ yield. The typically 20-fold increase in the insulin-to-4HCCA yield ratio, generated by water exposure of the samples, provides the explanation for the high yield ratios observed previously with water-treated samples. Spraying with water or repeated exposure to water droplets caused a pronounced degradation of the insulin parent yields in combination with an increasing appearance of signals due to the B- and A-chains of insulin. To clarify the issue of surface segregation, a few samples were prepared by spraying acetone-diluted solutions of insulin on previously deposited layers of 4HCCA. Whereas the insulin yields from as-prepared samples were rather low, the yields observed after water treatment were comparable with those observed with samples of insulin in 4HCCA. The results suggest that a large amount of insulin is present at the surface of samples prepared from liquid mixtures of insulin in 4HCCA. With both methods of sample preparation, however, high secondary ion yields of insulin were only obtained after exposure of the samples to water. The chemical changes responsible for this beneficial effect still need to be identified.