David Balgoma

Application of ’omics technologies to biomarker discovery in inflammatory lung diseases

Inflammatory lung diseases are highly complex in respect of pathogenesis and relationships between inflammation, clinical disease and response to treatment. Sophisticated large-scale analytical methods to quantify gene expression (transcriptomics), proteins (proteomics), lipids (lipidomics) and metabolites (metabolomics) in the lungs, blood and urine are now available to identify biomarkers that define disease in terms of combined clinical, physiological and patho-biological abnormalities. The aspiration is that these approaches will improve diagnosis, i.e. define pathological phenotypes, and facilitate the monitoring of disease and therapy, and also, unravel underlying molecular pathways. Biomarker studies can either select predefined biomarker(s) measured by specific methods or apply an “unbiased” approach involving detection platforms that are indiscriminate in focus. This article reviews the technologies presently available to study biomarkers of lung disease within the ’omics field. The contributions of the individual ’omics analytical platforms to the field of respiratory diseases are summarised, with the goal of providing background on their respective abilities to contribute to systems medicine-based studies of lung disease. Summary of the application of ’omics-based analytical platforms for biomarker discovery in inflammatory lung diseases http://ow.ly/mjGGc

Lipid Mediator Profiling in Pulmonary Disease

Oxylipins (e.g. eicosanoids) are endogenous signaling molecules that are formed from fatty acids by mono- or dioxygenase-catalyzed oxygenation and have been shown to play an important role in pathophysiological processes in the lung. These lipid mediators have been extensively studied for their role in inflammation in a broad swathe of respiratory diseases including asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis and extrinsic allergic alveolitis. Traditional efforts have employed analytical methods (e.g. radio- and enzyme-immunoassay techniques) capable of measuring a limited number of compounds simultaneously. The advent of the omics technologies is changing this approach and methods are being developed for the quantification of small molecules (i.e. metabolomics) as well as lipid-focused efforts (i.e. lipidomics). This review examines in detail the breadth of oxylipins and their biological activity in the respiratory system. In addition, the state-of-the-art methodology in profiling of oxylipins via mass spectrometry is summarized including sample work-up and data processing. These methods will greatly increase our ability to probe oxylipin biology and examine for cross-talk between biological pathways as well as specific compartments in the body. These new data will increase our insight into disease processes and have great potential to identify new biomarkers for disease diagnosis as well as novel therapeutic targets.

Quantification of lipid mediator metabolites in human urine from asthma patients by electrospray ionization mass spectrometry: controlling matrix effects.

Eicosanoids (e.g., prostaglandins and leukotrienes) are inflammatory signaling molecules that are metabolized and excreted in urine. The quantification of eicosanoid metabolites in human urine has been demonstrated to provide insight into the inflammatory and oxidative stress status of the individual. However, urine is a complex matrix that can exhibit profound matrix effects for quantification via liquid chromatography coupled to mass spectrometry (LC-MS/MS). This phenomenon can lead to impairment and biasing of results, because the sample background is dependent on the fluid intake and water-salt balance. Herein we describe an analytical methodology to address these limitations via the normalization of extracted urine volume by the ratio of absorbance at 300 nm to an optimized reference material. The platform is composed of 4 LC-MS/MS methods that collectively quantify 26 lipid mediators and their metabolites, with on-column limits of detection between 0.55 and 15 fmol. Prior to optimization, internal standards exhibited strong matrix effects with up to 50% loss of signal. Notably, the accuracy of exact deuterated structural analogues was found to vary based upon the number of incorporated deteurium. The platform was used to analyze urine from 16 atopic asthmatics under allergen provocation, showing increases in metabolites of prostaglandin D2, cysteinyl leukotrienes, and isoprostanes following the challenge. This method presents a functional and reproducible approach to addressing urine-specific matrix effects that can be readily formatted for quantifying large numbers of samples.

Linoleic acid-derived lipid mediators increase in a female-dominated subphenotype of COPD.

Chronic obstructive pulmonary disease (COPD) is a leading cause of mortality; however, the role of inflammatory mediators in its pathobiology remains unclear. The aim of this study was to investigate the influence of gender in COPD on lipid mediator levels. Bronchoalveolar lavage fluid (BALF) and serum were obtained from healthy never-smokers, smokers and COPD patients (Global Initiative for Chronic Obstructive Lung Disease stage I–II/A–B) (n=114). 94 lipid mediators derived from the cytochrome-P450, lipoxygenase, and cyclooxygenase pathways were analysed by liquid chromatography-mass spectrometry. Multivariate modelling identified a 9-lipid panel in BALF that classified female smokers with COPD from healthy female smokers (p=6×10−6). No differences were observed for the corresponding male population (p=1.0). These findings were replicated in an independent cohort with 92% accuracy (p=0.005). The strongest drivers were the cytochrome P450-derived epoxide products of linoleic acid (leukotoxins) and their corresponding soluble epoxide hydrolase (sEH)-derived products (leukotoxin-diols). These species correlated with lung function (r=0.87; p=0.0009) and mRNA levels of enzymes putatively involved in their biosynthesis (r=0.96; p=0.003). Leukotoxin levels correlated with goblet cell abundance (r=0.72; p=0.028). These findings suggest a mechanism by which goblet cell-associated cytochrome-P450 and sEH activity produce elevated leukotoxin-diol levels, which play a putative role in the clinical manifestations of COPD in a female-dominated disease sub-phenotype. Linoleic acid-derived lipids in BALF may indicate the transition from healthy smoker to COPD in a female subphenotype http://ow.ly/XowqN

Quantitative metabolic profiling of lipid mediators

Lipids are heterogeneous biological molecules that possess multiple physiological roles including cell structure, homeostasis, and restoration of tissue functionality during and after inflammation. Lipid metabolism constitutes a network of pathways that are related at multiple biosynthetic hubs. Disregulation of lipid metabolism can lead to pathophysiological effects and multiple lipid mediators have been described to be involved in physiological processes, (e.g. inflammation). Accordingly, a thorough description of these pathways may shed light on putative relations in multiple complex diseases, including chronic obstructive pulmonary disease, asthma, Alzheimers disease, multiple sclerosis, obesity, and cancer. Due to the structural complexity of lipids and the low abundance of many lipid mediators, mass spectrometry is the most commonly employed method for analysis. However, multiple challenges remain in the efforts to analyze every lipid subfamily. In this review, the biological role of sphingolipids, glycerolipids, oxylipins (e.g. eicosanoids), endocannabinoids, and N-acylethanolamines in relation to health and disease and the state-of-the-art analyses are summarized. The characterization and understanding of these pathways will increase our ability to examine for interrelations among lipid pathways and improve the knowledge of biological mechanisms in health and disease.

The Effect of Omega-3 Fatty Acids on Bronchial Hyperresponsiveness, Sputum Eosinophilia, and Mast Cell Mediators in Asthma

BACKGROUND Omega-3 fatty acid supplements have been reported to inhibit exercise-induced bronchoconstriction (EIB). It has not been determined whether omega-3 supplements inhibit airway sensitivity to inhaled mannitol, a test for bronchial hyperresponsiveness (BHR) and model for EIB in people with mild to moderate asthma. METHODS In a double-blind, crossover trial, subjects with asthma who had BHR to inhaled mannitol (n = 23; 14 men; mean age, 28 years; one-half taking regular inhaled corticosteroids) were randomized to omega-3 supplements (4.0 g/d eicosapentaenoic acid and 2.0 g/d docosahexaenoic acid) or matching placebo for 3 weeks separated by a 3-week washout. The primary outcome was the provoking dose of mannitol (mg) to cause a 15% fall in FEV1 (PD15). Secondary outcomes were sputum eosinophil count, spirometry, Asthma Control Questionnaire (ACQ) score, serum triacylglyceride level, and lipid mediator profile in urine and serum. RESULTS PD15 (geometric mean, 95% CI) to mannitol following supplementation with omega-3s (78 mg, 51-119 mg) was not different from placebo (88 mg, 56-139 mg, P = .5). There were no changes in sputum eosinophils (mean ± SD) in a subgroup of 11 subjects (omega-3, 8.4% ± 8.2%; placebo, 7.8% ± 11.8%; P = .9). At the end of each treatment period, there were no differences in FEV1 % predicted (omega-3, 85% ± 13%; placebo, 84% ± 11%; P = .9) or ACQ score (omega-3, 1.1% ± 0.5%; placebo, 1.1% ± 0.5%; P = .9) (n = 23). Omega-3s caused significant lowering of blood triglyceride levels and expected shifts in serum fatty acids and eicosanoid metabolites, confirming adherence to the supplements; however, no changes were observed in urinary mast cell mediators. CONCLUSIONS Three weeks of omega-3 supplements does not improve BHR to mannitol, decrease sputum eosinophil counts, or inhibit urinary excretion of mast cell mediators in people with mild to moderate asthma, indicating that dietary omega-3 supplementation is not useful in the short-term treatment of asthma. TRIAL REGISTRY ClinicalTrials.gov; No.: NCT00526357; URL: www.clinicaltrials.gov.