Statistical and bioinformatical methods to differentiate chronic obstructive pulmonary disease (COPD) including lung cancer from healthy control by breath analysis using ion mobility spectrometry

Human breath analysis is a powerful and especially a non-invasive technique for the monitoring and hopefully also for the diagnosis of respiratory diseases, including chronic obstructive pulmonary disease (COPD). The exhaled breath of 95 patients suffering COPD and of 35 healthy controls was investigated using an Ion Mobility Spectrometer (IMS) coupled to a Multi-Capillary Column (MCC) without any pre-separation or pre-enrichment. Starting with the results from a Mann–Whitney-Wilcoxon rank sum test to find analytes with the highest potential with respect to differentiation, box and whisker plots, metabolic maps and probability charts were introduced and compared. In addition, the sensitivity, specificity, positive and negative predictive values and the accuracy of the relation were also summarized. The findings were compared to the results of a principal component analysis. Finally, decision trees were introduced to visualize the interdependencies between the analytes and the classifications. The application of these biostatistical methods with simultaneous inclusion of several VOCs for disease classification by ion mobility spectrometry of human breath will provide much more information than using single peaks and single concentration dependencies for disease classification and discrimination of various groups. Towards the future application of potential biomarkers for clinical diagnostic procedures, complex analytical methods, such as ion mobility spectrometry, need statistical and bioinformatical tools which are simple in application, visualize the results and support decisions on the basis of the data obtained from measurements of analytes in exhaled human breath.     

Differential scanning calorimetry (DSC) of blood serum in chronic obstructive pulmonary disease (COPD)

Chronic obstructive pulmonary disease (COPD) is a major global health challenge with a gloom perspective of being one of the big three cause of death by 2020. No reliable/reproducible biomarker has been identified so far to match the clinically-based staging system (GOLD). Blood samples of 30 subjects divided into 6 groups (no-COPD/-smoker, no-COPD/non-smoker, COPD I, COPD II, COPD III, COPD IV) with 5 patients in each were tested by differential scanning calorimetry. There is a clear 15.4 % difference between the heat flow maxima measured when no-COPD subjects were compared in accordance to their smoking/non-smoking status. Odds ratio of different heat flow in actively smoking COPD patients in stage IV and stage I was 1.61. A reverse tendency is detected in the relevant non-smoking COPD groups. The differences are inconsistent in intermediate stages (COPD II and III). DSC seems to be an applicable and objective method for monitoring nicotine abuse. There is a chance to detect specific typology of thermokinetic patterns in the two extremes of COPD (I vs. IV). Further studies with increased sample size are needed to allow calculations on specificity/sensitivity/positive and negative predictive value of enthalpies and heat flow maximums. The first clinically relevant blood-based COPD marker on the intravascular side of the alveo-capillary screen is demonstrated by our pilot study.     

Differentiation of chronic obstructive pulmonary disease (COPD) including lung cancer from healthy control group by breath analysis using ion mobility spectrometry

Non-invasive methods with potential for diagnosis of lung diseases gain increasing interest. Within the present study the exhaled breath of 132 persons (97 Chronic obstructive pulmonary disease (COPD) patients [35 COPD without lung cancer, 62 COPD with lung cancer] and 35 healthy volunteers) was investigated using an Ion Mobility Spectrometer (IMS) coupled to a Multi-Capillary Column (MCC) without any pre-separation or pre-enrichment. One hundred four different peaks were considered within the IMS-Chromatograms of the 10 mL breath samples of both groups. A principal component analysis (PCA) of these 104 peaks identified a single analyte, that allowed a separation of the healthy persons and the COPD patients (with and without lung cancer). The sensitivity obtained was 60%, the specificity 91%, the positive predictive value 95%. The peak was characterized as cyclohexanone (CAS 108-94-1). Subsequent studies must validate the identity of the peak used for separation of the two groups with a greater population and external standards. Breath gas analysis using ion mobility spectrometry offers a chance of separating healthy persons and COPD patients using a single analyte at a defined concentration.     

Detection of volatile organic compounds (VOCs) in exhaled breath of patients with chronic obstructive pulmonary disease (COPD) by ion mobility spectrometry

COPD is a disease characterised by a chronic inflammation of the airways and a not fully reversible airway obstruction. The spirometry is considered as gold-standard to diagnose the disease and to grade its severity. In this study we used the methodology of Ion Mobility Spectometry in order to detect Volatile Organic Compounds (VOCs) in exhaled breath of patients with COPD. The purpose of this study was to investigate if the VOCs detected in patients with COPD were different from the VOCs detected in exhaled breath of healthy controls. 13 COPD patients and 33 healthy controls were included in the study. Breath samples were collected via a side-steam Teflon tube and directly measured by an ion mobility spectrometer coupled to a multi capillary column (MCC/IMS). One peak was identified only in the patients group compared to the healthy control group. Consequently, the analysis of exhaled breath could be a useful tool to diagnose COPD.     

Monoclonal antibody proteomics: discovery and prevalidation of chronic obstructive pulmonary disease biomarkers in a single step

We define mAb proteomics as the global generation of disease specific antibodies that permit mass screening of biomarkers. An integrated, high-throughput, disease-specific mAb-based biomarker discovery platform has been developed. The approach readily provided new biomarker leads with the focus on large-scale discovery and production of mAb-based, disease-specific clinical assay candidates. The outcome of the biomarker discovery process was a highly specific and sensitive assay, applicable for testing of clinical validation paradigms, like response to treatment or correlation with other clinical parameters. In contrast to MS-based or systems biology-based strategies, our process produced prevalidated clinical assays as the outcome of the discovery process. By re-engineering the biomarker discovery paradigm, the encouraging results presented in this paper clearly demonstrate the efficiency of the mAb proteomics approach, and set the grounds for the next steps of studies, namely, the hunt for candidate biomarkers that respond to drug treatment.     

Polymorphisms in the interleukin-8 gene in patients with chronic obstructive pulmonary disease

Airway inflammation is the main pathophysiological feature of patients with chronic obstructive pulmonary disease (COPD). Interleukin-8 (IL-8) is a potent chemoattractant for neutrophils and eosinophils. Increased IL-8 levels were observed in bronchoalveolar lavage (BAL) and induced sputum in patients with COPD. To evaluate the role of the IL-8 gene, we genotyped blood samples of 122 COPD-patients and 385 healthy controls for a known polymorphism in the promoter region (-251 A/T) of the IL-8 gene. Additionally, we screened the coding region for further polymorphisms by SSCP analyses. Comparison of the allele and genotype frequencies among each group revealed no significant differences between patients and controls. Although IL-8 plays an important role in the chemotaxis of inflammatory cells, the polymorphisms investigated here do not seem to be involved in the genetic predisposition to COPD.     

A promotor polymorphism in the Interleukin 11 gene is associated with chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a multifactorial disorder characterized by irreversible airflow obstruction due to chronic inflammation. Hence, the gene encoding the anti-inflammatory cytokine IL 11 is a good candidate for being involved in the genetic predisposition to COPD. In order to evaluate the role of the Interleukin 11 (IL 11) gene in the genetic predisposition for COPD, a dinucleotide microsatellite polymorphism in the promoter region has been genotyped in 153 patients with COPD (including 25 non-smokers) and 463 healthy controls. Frequencies of the IL 11.A2 microsatellite allele and of IL 11.A2 homozygous individuals were significantly decreased among the patients with COPD (p < 0.012 and p < 0.022, respectively) as compared to controls. Both frequencies were even more drastically reduced among the nonsmoking patients. Tight linkage of this microsatellite allele with another polymorphism in the promotor region was established. Altered expression of IL 11 may be involved in the genetic predisposition to COPD.     

Ultrafast gas chromatography coupled to electronic nose to identify volatile biomarkers in exhaled breath from chronic obstructive pulmonary disease patients: A pilot study

An analytical method to identify volatile organic compounds (VOCs) in the exhaled breath from patients with a diagnosis of chronic obstructive pulmonary disease (COPD) using a ultrafast gas chromatography system equipped with an electronic nose detector (FGC eNose) has been developed. A prospective study was performed in 23 COPD patients and 33 healthy volunteers; exhalation breathing tests were performed with Tedlar bags. Each sample was analyzed by FCG eNose and the identification of VOCs was based on the Kovats index. Raw data were reduced by principal component analysis (PCA) and canonical discriminant analysis [canonical analysis of principal coordinates (CAP)]. The FCG eNose technology was able to identify 17 VOCs that distinguish COPD patients from healthy volunteers. At all stages of PCA and CAP the discrimination between groups was obvious. Chemical prints were correctly classified up to 82.2%, and were matched with 78.9% of the VOCs detected in the exhaled breath samples. Receiver operating characteristic curve analysis indicated the sensitivity and specificity to be 96% and 91%, respectively. This pilot study demonstrates that FGC eNose is a useful tool to identify VOCs as biomarkers in exhaled breath from COPD patients. Further studies should be performed to enhance the clinical relevance of this quick and ease methodology for COPD diagnosis.     

Synergistic cycles of protease activity and inflammation via PPARγ degradation in chronic obstructive pulmonary disease

Inflammation, oxidative stress, and protease–antiprotease imbalance have been suggested to be a pathogenic triad in chronic obstructive pulmonary disease (COPD). However, it is not clear how proteases interact with components of inflammatory pathways. Therefore, this study aimed to evaluate the effect of neutrophil elastase (NE) on lipopolysaccharide (LPS)-induced interleukin 8 (IL-8) production and determine the molecular mechanism in human bronchial epithelial cells (HBECs). Immortalized bronchial epithelial cells and primary HBECs were used to investigate the impact of NE on LPS-induced IL-8 production. The molecular mechanism by which NE modulated LPS-induced IL-8 production was confirmed in elastase-treated C57BL/6 mice and primary HBECs obtained from COPD patients and healthy controls. The results showed that NE treatment synergistically augmented LPS-induced IL-8 production in both immortalized bronchial epithelial cells and primary HBECs. NE partially degraded peroxisome proliferator-activated receptor gamma (PPARγ), which is known to regulate IL-8 production in the nucleus. Treatment with a PPARγ agonist and overexpression of PPARγ reversed the NE-induced synergistic increase in LPS-induced IL-8 production. Moreover, PPARγ levels were lower in lung homogenates and lung epithelial cells from elastase-treated mice than in those from saline-treated mice. In accordance with the findings in mice, PPARγ levels were lower in primary HBECs from COPD patients than in those from healthy never-smokers or healthy smokers. In conclusion, a vicious cycle of mutual augmentation of protease activity and inflammation resulting from PPARγ degradation plays a role in the pathogenesis of COPD.Subject terms: Inflammation, Cytokines     

Magnetic systems with mixed carboxylate and azide bridges: slow relaxation in Co(II) metamagnet and spin frustration in Mn(II) compound

Two coordination polymers formulated as [{[Co(2)(L)(N(3))(4)]·2DMF}(n) (1) and [Mn(2)(L)(H(2)O)(0.5)(N(3))(8)](n) (2) (L = 1,4-bis(4-carboxylatopyridinium-1-methyl)benzene) were synthesized and structurally and magnetically characterized. In compound 1, the anionic uniform Co(II) chains with mixed (μ-EO-N(3))(2)(μ-COO) triple bridges (EO = end-on) are cross-linked by the cationic bis(pyridinium) spacers to generate 2D coordination layers. It was demonstrated that the triple bridges mediate ferromagnetic coupling and that the compound represents a new example of the rare systems exhibiting the coexistence of antiferromagnetic ordering, metamagnetism, and slow magnetic dynamics. Compound 2 features the magnetic Δ-chain formed from isosceles triangular units with single μ-EE-N(3) and double (μ-EO-N(3))(μ-COO) bridges (EE = end-to-end). The Δ-chains are interlinked by long organic ligands into a 3D framework with novel net topology and 3-fold interpenetration. The magnetic properties of 2 indicate the presence of spin frustration characteristic of Δ-chains with antiferromagnetic interactions.     

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the estimation of amlodipine in human plasma

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the estimation of amlodipine in human plasma. Amlodipine was extracted from human plasma by using a solid-phase extraction technique. Imipramine was used as the internal standard. A Hypersil BDS C18 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The method involves a rapid solid-phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables detection at sub-nanogram levels. The proposed method has been validated for a linear range of 0.1-10.0 ng/mL with correlation coefficient >or=0.9990. The intrarun and interrun precision and accuracy were within 10.0%. The overall recovery for amlodipine was 63.67%. Total run time was 3.2 min only.     

Metamagnetism and slow magnetic dynamics in an antiferromagnet composed of cobalt(II) chains with mixed azide-carboxylate bridges

A novel 2D metamagnetic compound composed of ferromagnetic Co(II) chains exhibits the intrinsic slow dynamics of single-chain magnets in the antiferromagnetic ordered phase and in the field-induced ferromagnetic phase.     

Combining restricted access material (RAM) and turbulent flow for the rapid on-line extraction of the cyclooxygenase-2 inhibitor rofecoxib in plasma samples

Restricted access material (RAM) has been used in the packing of a solid-phase extraction (SPE) column for on-line extractions under turbulent flow conditions. The bio-compatible RAM material works by the principle of size exclusion in addition to conventional reversed-phase chromatography, thereby allowing the extraction and preconcentration of small analyte molecules from biological samples such as plasma. Using small column dimensions (0.76 mm x 50 mm) and a consequently high linear velocity, turbulent flow was achieved during online sample extractions. The improved mass-transfer rate characteristic of turbulent flow allows fast sample cleanup without decreased extraction efficiency. The novel use of the RAM column, connected upstream to a C18 monolithic column, allowed the direct injection, extraction, separation, and MS/MS detection of plasma samples spiked with rofecoxib in a span of 5 min. Calibration curves obtained using this RAM turbulent flow coupled column method showed good linearity (R2 > 0.99) and reproducibility (%RSD < or = 7%). The lower limit of quantitation of rofecoxib in plasma samples was found to be 40 ng/ml. The extraction method showed good recovery of rofecoxib from a plasma matrix with minimal signal loss and robustness after more than 200 plasma injections.     

HPLC determination of cis-diamminedichloroplatinum(II) in plasma and urine with UV detection and column-switching

A method for determining cis-diamminedichloroplatinum(II) (CDDP), an anticancer drug, in plasma and urine by HPLC with UV detection and column-switching has been developed. Typical conditions were as follows. An apparatus was composed of two columns, two pumps, a UV detector, a sample injector with a 100 microL loop, a switching valve, a column oven and a recorder. A Rheodyne model 7125 sample injector was used as the switching valve. A precolumn (4.6 mm ID x 25 cm) was packed with MCI GEL CK10S (a strong cation exchanger), and an analytical column (4.6 mm ID x 5 cm) was packed with MCI GEL CDR10 (a strong anion exchanger). Both columns were connected in series via the switching valve. The CDDP-containing fraction of the effluent from the precolumn was loaded to the analytical column by column-switching and the effluent from the analytical column was monitored at 210 nm. An eluent of 0.3 M sodium dihydrogen phosphate was pumped at a flow rate of 1 mL/min and the columns were maintained at 40 degrees C. CDDP was eluted at about 11 min and the identity of the peak of CDDP on the chromatogram was confirmed by its 3-dimensional chromatogram and analysis of platinum in the column effluent. Under the conditions described above, a linear relationship was obtained between peak height and concentration of CDDP up to 100 microM. Correlation efficients were 0.998 for plasma and 0.999 for urine. The detection limit was 0.1 microM for CDDP in both plasma and urine (S/N = 3,0.005 AUFS). The reproducibility was within 3% for 10 determinations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interplay between kinetically slow thermal spin-crossover and metastable high-spin state relaxation in an iron(II) complex with similar T1/2 and T(LIESST)

This paper describes the first material to show the well-known light-induced excited spin-state trapping (LIESST) effect, the metastable excited state of which relaxes at a temperature approaching its thermal spin-crossover. Cooling polycrystalline [FeL(2)][BF(4)](2).x H(2)O (L=2,6-bis[3-methylpyrazol-1-yl]pyridine; x=0-1/3) at 1 K min(-1) leads to a cooperative spin transition, taking place in two steps centered at 147 and 105 K, that is only 54 % complete by magnetic susceptibility. Annealing the sample at 100 K for 2 h results in a slow decrease in chi(M)T to zero, showing that the remainder of the spin-crossover can proceed, but is kinetically slow. The crystalline high- and fully low-spin phases of [FeL(2)][BF(4)](2).x H(2)O are isostructural (C2/c, Z=8), but the spin-crossover proceeds via a mixed-spin intermediate phase that has a triple unit cell (C2/c, Z=24). The water content of the crystals is slowly lost on exposure to air without causing decomposition. However, the high-spin/mixed-spin transition in the crystal proceeds at 110+/-20 K when x=1/3 and 155+/-5 K when x=0, which correspond to the two spin-crossover steps seen in the bulk material. The high-spin state of the compound is generated quantitatively by irradiation of the low-spin or the mixed-spin phase at 10 K, and in approximately 70 % yield by rapidly quenching the sample to 10 K. This metastable high-spin state relaxes back to the low-spin ground state at 87+/-1 K in one, not two, steps, and without passing through the intermediate phase. This implies that thermal spin-crossover and thermally activated high-spin-low-spin relaxation in this material become decoupled, thus avoiding the physical impossibility of T(LIESST) being greater than T(1/2).     

Automated analysis of terbutaline (BricanylR) in human plasma with liquid chromatography and electrochemical detection using column-switching (multidimensional chromatography)

Summary An automated method based on liquid chromatography has been developed for the determination of terbutaline in human plasma in the range of 5–50 pmole·ml^–1. The necessary sensitivity and selectivity was obtained by using electrochemical detection and a microprocessor-controlled column switching system. A combination of three columns was used: a C₈ type for pre-separation, a C₁₈ type for trapping and, for final separation, a strongly acidic ion exchanger. The accuracy of the method was examined by comparison with a method based on gas chromatography — mass spectrometry. The overall precision was ±3.5% and ±2.2% respectively at 5 and 50 pmole·ml^–1. The total absolute recovery for terbutaline and internal standard at the above concentration levels were in the range 85–106%.Presented at the 14th International Symposium on Chromatography London, September, 1982