Darryl J. Adamko

Metabolomic profiling of asthma: diagnostic utility of urine nuclear magnetic resonance spectroscopy.

BACKGROUND The ability to diagnose and monitor asthma on the basis of noninvasive measurements of airway cellular dysfunction is difficult in the typical clinical setting. OBJECTIVE Metabolomics is the study of molecules created by cellular metabolic pathways. We hypothesized that the metabolic activity of children with asthma would differ from healthy children without asthma. Furthermore, children having an asthma exacerbation would be different compared with children with stable asthma in outpatient clinics. Finally, we hypothesized that (1)H-nuclear magnetic resonance (NMR) would measure such differences using urine samples, one of the least invasive forms of biofluid sampling. METHODS Children (135 total, ages 4-16 years) were enrolled, having met the criteria of healthy controls (C), stable asthma in the outpatient clinic (AO), or unstable asthma in the emergency department (AED). Partial least squares discriminant analysis was performed on the NMR data to create models of separation (70 metabolites were measured/urine sample). Some NMR data were withheld from modeling to be run blindly to determine possible diagnostic accuracy. RESULTS On the basis of the model of AO versus C, 31 of 33 AO samples were correctly diagnosed with asthma (94% accuracy). Only 1 of 20 C samples was incorrectly labeled as asthma (5% misclassification). On the basis of the AO versus AED model, 31 of the 33 AO samples were correctly diagnosed as outpatient asthma (94% accurate). CONCLUSION This is the first report suggesting that (1)H-NMR analysis of human urine samples has the potential to be a useful clinical tool for physicians treating asthma.

The rise of the phoenix: the expanding role of the eosinophil in health and disease

We have entered a new phase in the evolution of our understanding of the role of the eosinophil with a greater appreciation of novel potential functions that may be ascribed to this enigmatic cell type. This review not only provides an update to our current understanding of the various immunobiological roles for the eosinophil, but also attracts attention to some novel observations predicting functions beyond its putative effector role. These observations include the intriguing possibility that the eosinophil may posses the capacity to regulate the immune and inflammatory responses in diseases such as asthma.

Pneumococcal Pneumonia: Potential for Diagnosis through a Urinary Metabolic Profile

Pneumonia, an infection of the lower respiratory tract, is caused by any of a number of different microbial organisms including bacteria, viruses, fungi, and parasites. Community-acquired pneumonia (CAP) causes a significant number of deaths worldwide, and is the sixth leading cause of death in the United States. However, the pathogen(s) responsible for CAP can be difficult to identify, often leading to delays in appropriate antimicrobial therapies. In the present study, we use nuclear magnetic resonance spectroscopy to quantitatively measure the profile of metabolites excreted in the urine of patients with pneumonia caused by Streptococcus pneumoniae and other microbes. We found that the urinary metabolomic profile for pneumococcal pneumonia was significantly different from the profiles for viral and other bacterial forms of pneumonia. These data demonstrate that urinary metabolomic profiles may be useful for the effective diagnosis of CAP.

Effects of inflammatory cells on neuronal M2 muscarinic receptor function in the lung

In the lungs, acetylcholine released from the parasympathetic nerves stimulates M3 muscarinic receptors on airway smooth muscle inducing contraction and bronchoconstriction. The amount of acetylcholine released from these nerves is limited locally by neuronal M2 muscarinic receptors. These neuronal receptors are dysfunctional in asthma and in animal models of asthma. Decreased M2 muscarinic receptor function results in increased release of acetylcholine and in airway hyperreactivity. Inflammation has long been associated with hyperreactivity and the role of inflammatory cells in loss of neuronal M2 receptor function has been examined. There are several different mechanisms for loss of neuronal M2 receptor function. These include blockade by endogenous antagonists such as eosinophil major basic protein, decreased expression of M2 receptors following infection with viruses or exposure to pro inflammatory cytokines such as gamma interferon. Finally, the affinity of acetylcholine for these receptors can be decreased by exposure to neuraminidase.

Metabolomic biomarkers in a model of asthma exacerbation: urine nuclear magnetic resonance.

RATIONALE Airway obstruction in patients with asthma is associated with airway dysfunction and inflammation. Objective measurements including sputum analysis can guide therapy, but this is often not possible in typical clinical settings. Metabolomics is the study of molecules generated by metabolic pathways. We hypothesize that airway dysfunction and inflammation in an animal model of asthma would produce unique patterns of urine metabolites measured by multivariate statistical analysis of high-resolution proton nuclear magnetic resonance ((1)H NMR) spectroscopy data. OBJECTIVES To develop a noninvasive means of monitoring asthma status by metabolomics and urine sampling. METHODS Five groups of guinea pigs were studied: control, control treated with dexamethasone, sensitized (ovalbumin, administered intraperitoneally), sensitized and challenged (ovalbumin, administered intraperitoneally, plus ovalbumin aerosol), and sensitized-challenged with dexamethasone. Airway hyperreactivity (AHR) to histamine (administered intravenously) and inflammation were measured. Multivariate statistical analysis of NMR spectra based on a library of known urine metabolites was performed by partial least-squares discriminant analysis. In addition, the raw NMR spectra exported as xy-trace data underwent linear discriminant analysis. MEASUREMENTS AND MAIN RESULTS Challenged guinea pigs developed AHR and increased inflammation compared with sensitized or control animals. Dexamethasone significantly improved AHR. Using concentration differences in metabolites, partial least-squares discriminant analysis could discriminate challenged animals with 90% accuracy. Using only three or four regions of the NMR spectra, linear discriminant analysis-based classification demonstrated 80-90% separation of the animal groups. CONCLUSIONS Urine metabolites correlate with airway dysfunction in an asthma model. Urine NMR analysis is a promising, noninvasive technique for monitoring asthma in humans.

Mass spectrometric based approaches in urine metabolomics and biomarker discovery

Urine metabolomics has recently emerged as a prominent field for the discovery of non-invasive biomarkers that can detect subtle metabolic discrepancies in response to a specific disease or therapeutic intervention. Urine, compared to other biofluids, is characterized by its ease of collection, richness in metabolites and its ability to reflect imbalances of all biochemical pathways within the body. Following urine collection for metabolomic analysis, samples must be immediately frozen to quench any biogenic and/or non-biogenic chemical reactions. According to the aim of the experiment; sample preparation can vary from simple procedures such as filtration to more specific extraction protocols such as liquid-liquid extraction. Due to the lack of comprehensive studies on urine metabolome stability, higher storage temperatures (i.e. 4°C) and repetitive freeze-thaw cycles should be avoided. To date, among all analytical techniques, mass spectrometry (MS) provides the best sensitivity, selectivity and identification capabilities to analyze the majority of the metabolite composition in the urine. Combined with the qualitative and quantitative capabilities of MS, and due to the continuous improvements in its related technologies (i.e. ultra high-performance liquid chromatography [UPLC] and hydrophilic interaction liquid chromatography [HILIC]), liquid chromatography (LC)-MS is unequivocally the most utilized and the most informative analytical tool employed in urine metabolomics. Furthermore, differential isotope tagging techniques has provided a solution to ion suppression from urine matrix thus allowing for quantitative analysis. In addition to LC-MS, other MS-based technologies have been utilized in urine metabolomics. These include direct injection (infusion)-MS, capillary electrophoresis-MS and gas chromatography-MS. In this article, the current progresses of different MS-based techniques in exploring the urine metabolome as well as the recent findings in providing potentially diagnostic urinary biomarkers are discussed.

Virus-induced eosinophil mediator release requires antigen-presenting and CD4+ T cells

BACKGROUND The most frequent trigger of asthma exacerbation is infection with common airway viruses; however, the precise mechanism regulating such severe reactions is not understood. The presence of airway eosinophil products is a unique feature detected in asthmatic airways. Using an animal model, we previously demonstrated that T cells play an important role in regulating an eosinophil-dependant pathway of virus-induced airway hyperreactivity. We hypothesize that human eosinophils respond to viruses, although only after interaction with T cells. OBJECTIVES We sought to determine whether eosinophils can respond to airway viruses in vitro and determine the mechanism of response. METHODS An in vitro coculture model of human eosinophils, antigen-presenting cells, and T cells was used with parainfluenza virus, respiratory syncytial virus, or rhinovirus. We measured release of eosinophil peroxidase (EPO) in concert with T-cell proliferation, cytokine release, and changes in T-cell phenotype. RESULTS The viruses induced release of EPO when coincubated in the presence of antigen-presenting cells (dendritic cells or macrophages) and T cells. Virus-mediated release was associated with proliferation of CD3(+)CD4(+) T cells and release of cytokines. UV inactivation of the virus did not prevent virus-induced EPO release or T-cell proliferation. Proliferating CD4(+) T cells show increased expression of CD25 and CD45RO. CD8(+) T cells were not activated. CONCLUSION Virus-induced EPO release can occur in the context of antigen presentation to CD4(+) T cells.

The eosinophil as a therapeutic target in asthma: beginning of the end, or end of the beginning?

A major goal in asthma therapy is to reduce or prevent the inflammatory response associated with bronchial hyperresponsiveness, reversible airway obstruction and airway remodelling. However, because of the complex nature of the disease, a single target for such an ideal therapeutic approach remains elusive. To ensure a more rational design of anti-asthma drugs, recent investigations have attempted to elucidate the roles of inflammatory cellular components in asthma. Such studies have shown that eosinophilic infiltration is a prominent feature in the pathophysiology of asthma. Nonetheless, the role of the eosinophil in asthma has been questioned following recent human studies investigating the efficacy of a novel therapeutic strategy targeted at eosinophils.

The Metabolomics of Asthma: Novel Diagnostic Potential

Asthma is one of the most common chronic illnesses, especially in children. Reaching the diagnosis of asthma and its management are more difficult than for other chronic illnesses. For example, asthma is a heterogeneous syndrome with many clinical classifications based on patient symptoms, lung function, and response to therapy. The symptoms and objective measurements of lung function, often used to guide therapy, are largely based on the inflammation of the airways. Because measuring airway dysfunction and inflammation in a typical clinical setting is difficult, it is often not done. Metabolomics is the study of small molecules generated from cellular metabolic activity. It is possible that the metabolic profile of a patient with a chronic illness such as asthma is different from that of a healthy patient or from a patient with another respiratory illness. Furthermore, if this metabolome could be measured, it might also vary with disease severity. The pattern of metabolites becomes the diagnostic representing the disease. This article outlines the more recent work that has been done to develop the metabolomic profile of asthma.

Identifying Hypoxia in a Newborn Piglet Model Using Urinary NMR Metabolomic Profiling

Establishing the severity of hypoxic insult during the delivery of a neonate is key step in the determining the type of therapy administered. While successful therapy is present, current methods for assessing hypoxic injuries in the neonate are limited. Urine Nuclear Magnetic Resonance (NMR) metabolomics allows for the rapid non-invasive assessment of a multitude breakdown products of physiological processes. In a newborn piglet model of hypoxia, we used NMR spectroscopy to determine the levels of metabolites in urine samples, which were correlated with physiological measurements. Using PLS-DA analysis, we identified 13 urinary metabolites that differentiated hypoxic versus nonhypoxic animals (1-methylnicotinamide, 2-oxoglutarate, alanine, asparagine, betaine, citrate, creatine, fumarate, hippurate, lactate, N-acetylglycine, N-carbamoyl-β-alanine, and valine). Using this metabolomic profile, we then were able to blindly identify hypoxic animals correctly 84% of the time compared to nonhypoxic controls. This was better than using physiologic measures alone. Metabolomic profiling of urine has potential for identifying neonates that have undergone episodes of hypoxia.