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.

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.

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.

Back pain in the emergency department: Pathological fracture following spinal manipulation.

Back pain is one of the most common presentations to the emergency department. Though case reports of patients presenting with increased back pain following chiropractic spinal manipulations are rare, we have identified a case rarely reported in the literature where a potential injury from chiropractic manipulation resulted in a diagnosis of multiple myeloma. We have reported a previously healthy 66-year-old male who presented with persistent lower back pain over 4 weeks. An initial evaluation with thoracolumbar radiographs revealed no significant findings. Following initial presentation to the family physician, the patient underwent three treatments of spinal manipulation from his local chiropractor, which resulted in worsening lower back pain. A re-examination and new radiographs in the hospital revealed multiple compression fractures and an underlying diagnosis of multiple myeloma. We have explored current literature examining the prevalence of lower back pain, as well as the incidence of spinal fracture following chiropractic manipulation, and have highlighted a potential complication from chiropractic manipulation in a patient with an undiagnosed underlying neoplastic disorder.

Monitoring asthma status.

PURPOSE: Asthma is a chronic disorder of the airways involving inflammation and airway hyper-reactivity. Clinical diagnosis and monitoring of asthma must incorporate the immunological, biochemical, and histological changes of a chronic disorder, while recognizing acute phenotypic changes in order to optimally tailor therapeutics to each individual. RECENT FINDINGS: Articles published within the previous 18 months are summarized in this article in order to present an up to date review of the latest findings regarding the monitoring of asthma. The articles encompass a wide array of specialties from basic research and histology to clinical medicine as well as community medicine and nursing. SUMMARY: Exciting new advancements in the monitoring of asthma continue to unfold. Potentially new diagnostic and monitoring tools are highlighted in this study. Continued investigations may enable a select few methodologies to reach clinical utility in the ongoing monitoring and treatment of patients with asthma.

Investigations of the effects of gender, diurnal variation, and age in human urinary metabolomic profiles.

Metabolomics may have the capacity to revolutionize disease diagnosis through the identification of scores of metabolites that vary during environmental, pathogenic, or toxicological insult. NMR spectroscopy has become one of the main tools for measuring these changes since an NMR spectrum can accurately identify metabolites and their concentrations. The predominant approach in analyzing NMR data has been through the technique of spectral binning. However, identification of spectral areas in an NMR spectrum is insufficient for diagnostic evaluation, since it is unknown whether areas of interest are strictly caused by metabolic changes or are simply artifacts. In this paper, we explore differences in gender, diurnal variation, and age in a human population. We use the example of gender differences to compare traditional spectral binning techniques (NMR spectral areas) to novel targeted profiling techniques (metabolites and their concentrations). We show that targeted profiling produces robust models, generates accurate metabolite concentration data, and provides data that can be used to help understand metabolic differences in a healthy population. Metabolites relating to mitochondrial energy metabolism were found to differentiate gender and age. Dietary components and some metabolites related to circadian rhythms were found to differentiate time of day urine collection. The mechanisms by which these differences arise will be key to the discovery of new diagnostic tests and new understandings of the mechanism of disease.

Kinetic studies of calcium and cardiac troponin I peptide binding to human cardiac troponin C using NMR spectroscopy.

Ca2+ and human cardiac troponin I (cTnI) peptide binding to human cardiac troponin C (cTnC) have been investigated with the use of 2D [1H,15N] HSQC NMR spectroscopy. The spectral intensity, chemical shift, and line-shape changes were analyzed to obtain the dissociation ( K(D)) and off-rate ( k(off)) constants at 30 degrees C. The results show that sites III and IV exhibit 100-fold higher Ca2+ affinity than site II ( K(D(III,IV)) approximately 0.2 microM, K(D(II)) approximately 20 microM), but site II is partially occupied before sites III and IV are saturated. The addition of the first two equivalents of Ca2+ saturates 90% of sites III and IV and 20% of site II. This suggests that the Ca2+ occupancy of all three sites may contribute to the Ca2+-dependent regulation in muscle contraction. We have determined a k(off) of 5000 s(-1) for site II Ca2+ dissociation at 30 degrees C. Such a rapid off-rate had not been previously measured. Three cTnI peptides, cTnI(34-71), cTnI(128-147), and cTnI(147-163), were titrated to Ca2+-saturated cTnC. In each case, the binding occurs with a 1:1 stoichiometry. The determined K(D) and k(off) values are 1 microM and 5 s(-1) for cTnI(34-71), 78+/-10 microM and 5000 s(-1) for cTnI(128-147), and 150+/-10 microM and 5000 s(-1) for cTnI(147-163), respectively. Thus, the dissociation of Ca2+ from site II and cTnI(128-147) and cTnI(147-163) from cTnC are rapid enough to be involved in the contraction/relaxation cycle of cardiac muscle, while that of cTnI(34-71) from cTnC may be too slow for this process.

NMR analysis of neutrophil activation in sputum samples from patients with cystic fibrosis.

Disorders of the respiratory system, such as cystic fibrosis (CF), involve the infiltration and activation of airway inflammatory cells, including neutrophils. This leads to the secretion of peroxidases, which react further with substrates in solution to produce oxidative metabolites, such as 3-chlorotyrosine. Elevated levels of modified tyrosine residues in the airways of patients with CF may be detectable by nuclear magnetic resonance (NMR) in correlation with inflammatory cell influx. In this study, high-resolution (500 MHz) 1H NMR was used to analyze the production of modified tyrosine residues resulting from in vitro stimulation of peripheral blood eosinophils and neutrophils, as well as in sputum samples from control subjects and patients with CF. Following in vitro stimulation, purified peripheral blood neutrophils generated 3-chlorotyrosine, while eosinophils produced predominantly 3-bromotyrosine and 3,5-dibromotyrosine. Chlorinated and brominated tyrosine residues were detected in sputum samples from patients with CF (N=7), but were not detected in the control group (N=9). Neutrophil counts in CF sputum correlated strongly with the presence of 3-chlorotyrosine (r2=0.869). Our findings indicate that neutrophil and eosinophil activation in CF is detectable by NMR. NMR may be a useful tool for the detection of biological markers of inflammatory processes in patient airways.