Profiling of Volatile Organic Compounds in Exhaled Breath As a Strategy to Find Early Predictive Signatures of Asthma in Children

Wheezing is one of the most common respiratory symptoms in preschool children under six years old. Currently, no tests are available that predict at early stage who will develop asthma and who will be a transient wheezer. Diagnostic tests of asthma are reliable in adults but the same tests are difficult to use in children, because they are invasive and require active cooperation of the patient. A non-invasive alternative is needed for children. Volatile Organic Compounds (VOCs) excreted in breath could yield such non-invasive and patient-friendly diagnostic. The aim of this study was to identify VOCs in the breath of preschool children (inclusion at age 2–4 years) that indicate preclinical asthma. For that purpose we analyzed the total array of exhaled VOCs with Gas Chromatography time of flight Mass Spectrometry of 252 children between 2 and 6 years of age. Breath samples were collected at multiple time points of each child. Each breath-o-gram contained between 300 and 500 VOCs; in total 3256 different compounds were identified across all samples. Using two multivariate methods, Random Forests and dissimilarity Partial Least Squares Discriminant Analysis, we were able to select a set of 17 VOCs which discriminated preschool asthmatic children from transient wheezing children. The correct prediction rate was equal to 80% in an independent test set. These VOCs are related to oxidative stress caused by inflammation in the lungs and consequently lipid peroxidation. In conclusion, we showed that VOCs in the exhaled breath predict the subsequent development of asthma which might guide early treatment.     

microRNA‐379 couples glucocorticoid hormones to dysfunctional lipid homeostasis

In mammals, glucocorticoids (GCs) and their intracellular receptor, the glucocorticoid receptor (GR), represent critical checkpoints in the endocrine control of energy homeostasis. Indeed, aberrant GC action is linked to severe metabolic stress conditions as seen in Cushing's syndrome, GC therapy and certain components of the Metabolic Syndrome, including obesity and insulin resistance. Here, we identify the hepatic induction of the mammalian conserved microRNA (miR)‐379/410 genomic cluster as a key component of GC/GR‐driven metabolic dysfunction. Particularly, miR‐379 was up‐regulated in mouse models of hyperglucocorticoidemia and obesity as well as human liver in a GC/GR‐dependent manner. Hepatocyte‐specific silencing of miR‐379 substantially reduced circulating very‐low‐density lipoprotein (VLDL)‐associated triglyceride (TG) levels in healthy mice and normalized aberrant lipid profiles in metabolically challenged animals, mediated through miR‐379 effects on key receptors in hepatic TG re‐uptake. As hepatic miR‐379 levels were also correlated with GC and TG levels in human obese patients, the identification of a GC/GR‐controlled miRNA cluster not only defines a novel layer of hormone‐dependent metabolic control but also paves the way to alternative miRNA‐based therapeutic approaches in metabolic dysfunction.     

Reaction mechanism and fragment ion structure determination of deprotonated small oligosaccharides, studied by negative ion fast atom bombardment (tandem) mass spectrometry

The negative ion mass spectrometric characteristics of a series of di- and trisaccharides and the tetrasaccharide stachyose have been studied using fast atom bombardment mass spectrometry. The molecular weight of the compounds can easily be derived from their mass spectra, which all show an abundant [M - H]- ion peak. The application of metastable ion and collisional activation techniques to selected pseudomolecular and fragment ions appears to be appropriate for the determination of the position and anomeric type of linkage in the molecules, and provides information concerning the monosaccharide units involved. Important fragmentation reactions have been traced and reaction mechanisms, supported by deuterium labelling experiments, are proposed. An experiment describing the application of the findings of this study to a glycosphingolipid molecule demonstrates its potential value for biological systems.     

Helicobacter pylori does not mediate the formation of carcinogenic N-nitrosamines

Background. Both N‐nitroso compounds and colonization with Helicobacter pylori represent known risk‐factors for the development of gastric cancer. Endogenous formation of N‐nitroso compounds is thought to occur predominantly in acidic environments such as the stomach. At neutral pH, bacteria can catalyze the formation of N‐nitroso compounds. Based on experiments with a noncarcinogenic N‐nitroso compound as end product, and using only a single H. pylori strain, it was recently reported that H. pylori only displays a low nitrosation capacity. As H. pylori is a highly diverse bacterial species, it is reasonable to question the generality of this finding. In this study, several genetically distinct H. pylori strains are tested for their capacity to form carcinogenic N‐nitrosamines. Materials and Methods. Bacteria were grown in the presence of 0–1000 µM morpholine and nitrite (in a 1 : 1 molar ratio), at pH 7, 5 and 3. Results. Incubation of Neisseria cinerea (positive control) with 500 µM morpholine and 500 µM nitrite, resulted in a significant increase in formation of N‐nitrosomorpholine, but there was no significant induction of N‐nitrosomorpholine formation by any of the H. pylori strains, at any of the three pH conditions. Conclusion. H. pylori does not induce formation of the carcinogenic N‐nitrosomorpholine in vitro. The previously reported weak nitrosation capacity of H. pylori is not sufficient to nitrosate the more difficultly nitrosatable morpholine. This probably also holds true for other secondary amines. These results imply that the increased incidence of gastric cancer formation that is associated with gastric colonization by H. pylori is unlikely to result from the direct induced formation of carcinogenic nitrosamines by H. pylori. However, this has to be further confirmed in in vivo studies.     

Adrenal Function in Females with Low Plasma HDL-C Due to Mutations in ABCA1 and LCAT

Introduction

Adrenal steroidogenesis is essential for human survival and depends on the availability of the precursor cholesterol. Male subjects with low plasma levels of high density lipoprotein (HDL) cholesterol are characterized by decreased adrenal function. Whether this is also the case in female subjects with low plasma HDL-C levels is unresolved to date.

Findings

15 female ATP binding cassette transporter AI (ABCAI) and 14 female lecithin-cholesterol acyltransferase (LCAT) were included in the study. HDL-C levels were 38% and 41% lower in ABCA1 and LCAT mutation carriers compared to controls, respectively. Urinary steroid excretion of 17-ketogenic steroids or 17-hydroxy corticosteroids did not differ between 15 female ABCA1 mutation carriers (p = 0.27 vs 0.30 respectively) and 30 matched normolipidemic controls or between 14 female LCAT mutation carriers and 28 matched normolipidemic controls (p = 0.10 and 0.14, respectively). Cosyntropin testing in an unselected subgroup of 8 ABCA1 mutation carriers and 3 LCAT mutation carriers did not reveal differences between carriers and controls.

Conclusion

Adrenal function in females with molecularly defined low HDL-C levels is not different from controls. The discrepancy with the finding of impaired steroidogenesis in males with molecularly defined low HDL-C levels underscores the importance of gender specific analyses in cholesterol-related research.     

Computational Screening of Tip and Stalk Cell Behavior Proposes a Role for Apelin Signaling in Sprout Progression

Angiogenesis involves the formation of new blood vessels by sprouting or splitting of existing blood vessels. During sprouting, a highly motile type of endothelial cell, called the tip cell, migrates from the blood vessels followed by stalk cells, an endothelial cell type that forms the body of the sprout. To get more insight into how tip cells contribute to angiogenesis, we extended an existing computational model of vascular network formation based on the cellular Potts model with tip and stalk differentiation, without making a priori assumptions about the differences between tip cells and stalk cells. To predict potential differences, we looked for parameter values that make tip cells (a) move to the sprout tip, and (b) change the morphology of the angiogenic networks. The screening predicted that if tip cells respond less effectively to an endothelial chemoattractant than stalk cells, they move to the tips of the sprouts, which impacts the morphology of the networks. A comparison of this model prediction with genes expressed differentially in tip and stalk cells revealed that the endothelial chemoattractant Apelin and its receptor APJ may match the model prediction. To test the model prediction we inhibited Apelin signaling in our model and in an in vitro model of angiogenic sprouting, and found that in both cases inhibition of Apelin or of its receptor APJ reduces sprouting. Based on the prediction of the computational model, we propose that the differential expression of Apelin and APJ yields a “self-generated” gradient mechanisms that accelerates the extension of the sprout.     

The effect of the trolox equivalent antioxidant capacity (TEAC) in plasma on the formation of 4-aminobiphenylhaemoglobin adducts in smokers

Smokers are exposed to a number of carcinogenic compounds including aromatic amines such as 4-aminobiphenyl. Antioxidants are thought to be involved in the defence against the damaging effect of such carcinogens. Recently it has been shown that plasma antioxidant status in smokers is diminished compared with non-smokers. In this study we investigated in 40 smokers whether the trolox equivalent antioxidant capacity (TEAC) in plasma could be quantitatively related to exposure to cigarette smoke. The biomarkers 4-aminobiphenylhaemoglobin (4-ABP-Hb) adduct and cotinine were determined as indices of cigarette smoke exposure. A correlation between 4-ABP-Hb adduct levels and plasma cotinine levels was found for the whole population studied, who smoked 4-70 cigarettes per day (n = 40, r² = 0.12, p = 0.03). A significant inverse relationship was found between TEAC and 4-ABP-Hb levels (n = 40, r² = 0.17, p = 0.008). Multiple regression analysis showed a strong relationship between 4-ABP-Hb levels and plasma TEAC and cotinine levels (n = 40, r² =0.29, p = 0.002). These findings provide strong evidence that the 4- ABP-Hb adduct represents a valuable biomarker of (internal) exposure to tobacco smoke, and also that the formation of this marker is dependent on the plasma antioxidant status. The multiple regression analysis results show that the measure of effect (4-ABP-Hb adduct formation) is largely determined by dose (cotinine) and protection (TEAC).     

Positive ion fast atom bombardment mass spectrometry of some small oligosaccharides

Positive ion fast atom bombardment mass spectrometry appears to be a very useful method for the determination of molecular weight and composition of underivatized di- and trisaccharides. Information on the identity of the monosaccharide units can be obtained from the metastable ion and collisional activation spectra of selected ions. The type of linkage between the monosaccharides is reflected in some spectral characteristics, but the differences are relatively small and do not always allow an unambiguous identification. The position of a fructose unit in a trisaccharide molecule is shown by the collisional activation spectra of the [M + H]+ ion, as an anhydrofructose molecule is easily eliminated from the ions in which fructose is in a terminal position.     

Mannosyl-β(1-4)-N-acetylglucosaminyl-β(1-N)-urea,a compound isolated from the urine of patients with β-mannosidosis

A previously unknown substance, mannosyl-(1–4)-N-acetylglucosaminyl-(1-N)-urea, has been isolated from the urine of patients with -mannosidosis in addition to the main metabolite mannosyl-(1–4)-N-acetylglucosamine. Structural investigation was carried out by fast atom bombardment mass spectrometry and high-resolution¹H-nuclear magnetic resonance spectroscopy at 500 MHz. It was postulated that the occurrence of this carbohydrate-urea conjugate in urine results mainly from urine handling.     

Defining adult asthma endotypes by clinical features and patterns of volatile organic compounds in exhaled air

Background

Several classifications of adult asthma patients using cluster analyses based on clinical and demographic information has resulted in clinical phenotypic clusters that do not address molecular mechanisms. Volatile organic compounds (VOC) in exhaled air are released during inflammation in response to oxidative stress as a result of activated leukocytes. VOC profiles in exhaled air could distinguish between asthma patients and healthy subjects. In this study, we aimed to classify new asthma endotypes by combining inflammatory mechanisms investigated by VOC profiles in exhaled air and clinical information of asthma patients.

Methods

Breath samples were analyzed for VOC profiles by gas chromatography–mass spectrometry from asthma patients (n = 195) and healthy controls (n = 40). A total of 945 determined compounds were subjected to discriminant analysis to find those that could discriminate healthy from asthmatic subjects. 2-step cluster analysis based on clinical information and VOCs in exhaled air were used to form asthma endotypes.

Results

We identified 16 VOCs, which could distinguish between healthy and asthma subjects with a sensitivity of 100% and a specificity of 91.1%. Cluster analysis based on VOCs in exhaled air and the clinical parameters FEV1, FEV1 change after 3 weeks of hospitalization, allergic sensitization, Junipers symptoms score and asthma medications resulted in the formation of 7 different asthma endotype clusters. We identified asthma clusters with different VOC profiles but similar clinical characteristics and endotypes with similar VOC profiles, but distinct clinical characteristics.

Conclusion

This study demonstrates that both, clinical presentation of asthma and inflammatory mechanisms in the airways should be considered for classification of asthma subtypes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0136-8) contains supplementary material, which is available to authorized users.