Lung cancer identification by the analysis of breath by means of an array of non-selective gas sensors

Previous finding shown that the composition of the breath of patients with lung cancer contains information that could be used to detect the disease. These volatiles are mainly alkanes and aromatic compounds. Sensor arrays technology (electronic nose) proved to be useful to screen samples characterised by different headspace composition. Here we investigated the possibility of using an electronic nose to check whether volatile compounds present in expired air may diagnose lung cancer. Breath samples were collected and immediately analysed by an electronic nose. A total of 60 individuals were involved in the experiment. 35 of them were affected by lung cancer, 18 individuals were measured as reference and nine were measured after the surgical therapy. Two individuals were measured twice, before and after the surgical therapy, for a total of 62 measurements. An electronic nose, composed by eight quartz microbalance (QMB) gas sensors, coated with different metalloporphyrins, was used. These sensors show a good sensitivity towards those compounds previously indicated as possible lung cancer markers in breath. The application of a 'partial least squares-discriminant analysis' (PLS-DA) found out a 100% of classification of lung cancer affected patients, 94% of reference was correctly classified. The class of post-surgery patients were correctly individuated in 44% of the cases, while the other samples were classified as healthy references. The alteration of breath composition induced by the presence of lung cancer was enough to allow a complete identification of the sample of diseased individuals. Extended studies are necessary to evaluate the resolution of the method, namely the stage at which the disease may be identified in order to use this instrument for early diagnosis.     

Collecting Protein Biomarkers in Breath Using Electret Filters: A Preliminary Method on New Technical Model and Human Study

Biomarkers in exhaled breath are useful for respiratory disease diagnosis in human volunteers. Conventional methods that collect non-volatile biomarkers, however, necessitate an extensive dilution and sanitation processes that lowers collection efficiencies and convenience of use. Electret filter emerged in recent decade to collect virus biomarkers in exhaled breath given its simplicity and effectiveness. To investigate the capability of electret filters to collect protein biomarkers, a model that consists of an atomizer that produces protein aerosol and an electret filter that collects albumin and carcinoembryonic antigen-a typical biomarker in lung cancer development- from the atomizer is developed. A device using electret filter as the collecting medium is designed to collect human albumin from exhaled breath of 6 volunteers. Comparison of the collecting ability between the electret filter method and other 2 reported methods is finally performed based on the amounts of albumin collected from human exhaled breath. In conclusion, a decreasing collection efficiency ranging from 17.6% to 2.3% for atomized albumin aerosol and 42% to 12.5% for atomized carcinoembryonic antigen particles is found; moreover, an optimum volume of sampling human exhaled breath ranging from 100 L to 200 L is also observed; finally, the self-designed collecting device shows a significantly better performance in collecting albumin from human exhaled breath than the exhaled breath condensate method (p<0.05) but is not significantly more effective than reported 3-stage impactor method (p>0.05). In summary, electret filters are potential in collecting non-volatile biomarkers in human exhaled breath not only because it was simpler, cheaper and easier to use than traditional methods but also for its better collecting performance.     

Determination of volatile organic compounds as biomarkers of lung cancer by SPME-GC-TOF/MS and chemometrics

A method for qualitative and quantitative the determination of concentrations volatile organic compounds (VOCs) in human breath samples using solid phase microextraction (SPME) and gas chromatography-time of flight-mass spectrometry (GC-TOF/MS) has been carried out. They are employed for the preconcentration, separation and analysis of biological samples. The technique to rapid determination compounds present in human air, at the level of parts per billion (ppb) is applied. This method was optimized and evaluated. It showed linear correlations ranging from 0.83 to 234.05 ppb, limit of detection in the range of 0.31 to 0.75 ppb and precision, expressed as the RSD, was less then 10.00%. The unique combination of statistical methods allowed reduce the number of compounds to significant ones only and indicate the potential way to find the biomarkers of the lung cancer. Presented an analytical and statistical methods for detection composition of exhaled air could be applied as a potential non-intrusive tool for screening of lung cancer.     

Biomarkers in breath condensate: a promising new non-invasive technique in free radical research

Oxidative stress is associated with a range of inflammatory lung diseases including asthma, adult respiratory distress syndrome, idiopathic pulmonary fibrosis, pneumonia, lung transplantation, chronic obstructive pulmonary disease, cystic fibrosis, bronchiectasis and lung cancer. Increased concentrations of reactive oxygen species (ROS) in the airways of such patients are reflected by elevated concentrations of oxidative stress markers in the breath, airways, lung tissue and blood. Traditionally, the measurement of these biomarkers has involved invasive procedures to procure the samples, or examine the compartments. As a consequence, there is a need for less invasive approaches to measure oxidative stress. Analysis of breath hydrocarbons has partly fulfilled this need, however only gas phase volatile constituents can be assessed by this approach. The collection of exhaled breath condensate (EBC) is a simple, non-invasive approach, which comprehensively samples the lower respiratory tract. It is currently used as a research and diagnostic tool in the free radical field, yielding information on redox disturbance and the degree and type of inflammation in the lung. With further technical developments, such an approach may ultimately have a role in the clinic, in helping to diagnose specific lung diseases. EBC can be exploited to assess a spectrum of potential biomarkers, thus generating a “finger print” characteristic of the disease. By assessing the nature of oxidative stress in this manner, the most appropriate therapy can be selected and the response to treatment monitored.     

ReviewBiomarkers in Breath Condensate: A promising New Non-invasive Technique in Free Radical Research

Oxidative stress is associated with a range of inflammatory lung diseases including asthma, adult respiratory distress syndrome, idiopathic pulmonary fibrosis, pneumonia, lung transplantation, chronic obstructive pulmonary disease, cystic fibrosis, bronchiectasis and lung cancer. Increased concentrations of reactive oxygen species (ROS) in the airways of such patients are reflected by elevated concentrations of oxidative stress markers in the breath, airways, lung tissue and blood. Traditionally, the measurement of these biomarkers has involved invasive procedures to procure the samples, or examine the compartments. As a consequence, there is a need for less invasive approaches to measure oxidative stress. Analysis of breath hydrocarbons has partly fulfilled this need, however only gas phase volatile constituents can be assessed by this approach. The collection of exhaled breath condensate (EBC) is a simple, non-invasive approach, which comprehensively samples the lower respiratory tract. It is currently used as a research and diagnostic tool in the free radical field, yielding information on redox disturbance and the degree and type of inflammation in the lung. With further technical developments, such an approach may ultimately have a role in the clinic, in helping to diagnose specific lung diseases. EBC can be exploited to assess a spectrum of potential biomarkers, thus generating a “finger print” characteristic of the disease. By assessing the nature of oxidative stress in this manner, the most appropriate therapy can be selected and the response to treatment monitored.     

Protein profiling in human sera for identification of potential lung cancer biomarkers using antibody microarray

To identify potential biomarkers of lung cancer (LC), profiling of proteins in sera obtained from healthy and LC patients was determined using an antibody microarray. Based on our previous study on mRNA expression profiles between patients with LC and healthy persons, 19 proteins of interest were selected as targets for fabrication of an antibody microarray. Antibody to each protein and five nonspecific control antibodies were spotted onto a hydrogel‐coated glass slide and used for profiling of proteins in sera of LC patients in a two‐color fluorescence assay. Forty‐eight human sera samples were analyzed, and expression profiling of proteins were represented by the internally normalized ratio method. Six proteins were distinctly down‐regulated in sera of LC patients; this observation was validated by Wilcoxon test, false discovery rate, and Western blotting. Blind test of other 32 human sera using the antibody microarray followed by hierarchical clustering analysis revealed an approximate sensitivity of 88%, specificity of 80%, and an accuracy of 84%, respectively, in classifying the sera, which supports the potential of the six identified proteins as biomarkers for the prognosis of lung cancer.     

Isolation of the crude oil degrading marine Acinetobacter sp. E11

The Acinetobacter sp. E11, isolated from Port Dickson Beach, Malaysia, was able to grow in media containing crude oil as the sole carbon and energy source. Substrate specificity studies showed that the bacterium exhibited substrate preference as growth was observed only in media containing aliphatic hydrocarbons, while aromatic and cyclic hydrocarbons inhibited growth. With the aliphatic hydrocarbons, growth was seen only in the long-chain alkanes tested (pentadecane, dodecane and hexadecane). No growth was recorded in the short-chain alkanes (pentane, hexane and heptane) tested. With complex hydrocarbons, only crude oil and 4T SHELL engine oil supported growth. No growth was observed in kerosene and PETRONAS gasoline. The isolate could grow in up to 10% and 20% [v/v] of the crude oil and alkanes tested, respectively. Among the long-chain alkanes tested, hexadecane was the most preferred, followed by pentadecane and dodecane. Nitrogen and phosphorous supplements were essential for growth and the best growth was achieved with 3% nitrogen/phosphorous additions. Microscopic observation revealed that the bacterium adhered to the hexadecane and crude oil droplets. GC analysis showed that the bacterium was able to degrade more than 60% of the hydrocarbons in the crude oil in 15 days at 37°C compared to the uninoculated media.     

Serum Calprotectin,CD26 and EGF to Establish a Panel for the Diagnosis of Lung Cancer

Lung cancer is the most lethal neoplasia, and an early diagnosis is the best way for improving survival. Symptomatic patients attending Pulmonary Services could be diagnosed with lung cancer earlier if high-risk individuals are promptly separated from healthy individuals and patients with benign respiratory pathologies. We searched for a convenient non-invasive serum test to define which patients should have more immediate clinical tests. Six cancer-associated molecules (HB-EGF, EGF, EGFR, sCD26, VEGF, and Calprotectin) were investigated in this study. Markers were measured in serum by specific ELISAs, in an unselected population that included 72 lung cancer patients of different histological types and 56 control subjects (healthy individuals and patients with benign pulmonary pathologies). Boosted regression and random forests analysis were conducted for the selection of the best candidate biomarkers. A remarkable discriminatory capacity was observed for EGF, sCD26, and especially for Calprotectin, these three molecules constituting a marker panel boasting a sensitivity of 83% and specificity of 87%, resulting in an associated misclassification rate of 15%. Finally, an algorithm derived by logistic regression and a nomogram allowed generating classification scores in terms of the risk of a patient of suffering lung cancer. In conclusion, we propose a non-invasive test to identify patients at high-risk for lung cancer from a non-selected population attending a Pulmonary Service. The efficacy of this three-marker panel must be tested in a larger population for lung cancer.     

Circadian Variation of the Human Metabolome Captured by Real-Time Breath Analysis

Circadian clocks play a significant role in the correct timing of physiological metabolism, and clock disruption might lead to pathological changes of metabolism. One interesting method to assess the current state of metabolism is metabolomics. Metabolomics tries to capture the entirety of small molecules, i.e. the building blocks of metabolism, in a given matrix, such as blood, saliva or urine. Using mass spectrometric approaches we and others have shown that a significant portion of the human metabolome in saliva and blood exhibits circadian modulation; independent of food intake or sleep/wake rhythms. Recent advances in mass spectrometry techniques have introduced completely non-invasive breathprinting; a method to instantaneously assess small metabolites in human breath. In this proof-of-principle study, we extend these findings about the impact of circadian clocks on metabolomics to exhaled breath. As previously established, our method allows for real-time analysis of a rich matrix during frequent non-invasive sampling. We sampled the breath of three healthy, non-smoking human volunteers in hourly intervals for 24 hours during total sleep deprivation, and found 111 features in the breath of all individuals, 36–49% of which showed significant circadian variation in at least one individual. Our data suggest that real-time mass spectrometric "breathprinting" has high potential to become a useful tool to understand circadian metabolism, and develop new biomarkers to easily and in real-time assess circadian clock phase and function in experimental and clinical settings.     

Determining the identity and roles of oil-metabolizing marine bacteria from the Thames estuary, UK

Crude oil is a complex mixture of different hydrocarbons. While diverse bacterial communities can degrade oil, the specific roles of individual members within such communities remain unclear. To identify the key bacterial taxa involved in aerobic degradation of specific hydrocarbons, microcosm experiments were established using seawater from Stanford le Hope, Thames estuary, UK, adjacent to a major oil refinery. In all microcosms, hydrocarbon degradation was significant within 10 weeks, ranging from > 99% of low-molecular-weight alkanes (C(10)-C(18)), 41-84% of high-molecular-weight alkanes (C(20)-C(32)) and pristane, and 32-88% of polycyclic aromatic hydrocarbons (PAHs). Analysis of 16S rRNA sequences from clone libraries and denaturing gradient gel electrophoresis (DGGE) indicated that, except when incubated with fluorene, PAH-degrading communities were dominated by Cycloclasticus. Moreover, PAH-degrading communities were distinct from those in microcosms containing alkanes. Degradation of the branched alkane, pristane, was carried out almost exclusively by Alcanivorax. Bacteria related to Thalassolituus oleivorans (99-100% identity) were the dominant known alkane degraders in n-alkane (C(12)-C(32)) microcosms, while Roseobacter-related bacteria were also consistently found in these microcosms. However, in contrast to previous studies, Thalassolituus, rather than Alcanivorax, was dominant in crude oil-enriched microcosms. The communities in n-decane microcosms differed from those in microcosms supplemented with less volatile alkanes, with a phylogenetically distinct species of Thalassolituus out-competing T. oleivorans. These data suggest that the diversity and importance of the genus Thalassolituus is greater than previously established. Overall, these experiments demonstrate how degradation of different petroleum hydrocarbons is partitioned between different bacterial taxa, which together as a community can remediate petroleum hydrocarbon-impacted estuarine environments.     

Is Pentane a Normal Constituent of Human Breath?

Breath analysis is a non-invasive method for investigation of the volatile compounds produced by humans. Pentane has often been taken as an indicator of lipid peroxidation. Our purpose in this study was to determine its normal concentration in the breath of healthy humans. Using a specific and sensitive gas chromatography-mass spectrometry technique pentane concentrations in breath were lower than 10 pmoles/1. The high levels of pentane found by some authors in healthy humans were probably due to the coelution of pentane with isoprene, a volatile hydrocarbon present in human breath.     

Is Pentane a Normal Constituent of Human Breath?

Breath analysis is a non-invasive method for investigation of the volatile compounds produced by humans. Pentane has often been taken as an indicator of lipid peroxidation. Our purpose in this study was to determine its normal concentration in the breath of healthy humans. Using a specific and sensitive gas chromatography-mass spectrometry technique pentane concentrations in breath were lower than 10 pmoles/1. The high levels of pentane found by some authors in healthy humans were probably due to the coelution of pentane with isoprene, a volatile hydrocarbon present in human breath.     

Investigation of volatile biomarkers in lung cancer blood using solid-phase microextraction and capillary gas chromatography-mass spectrometry

In the present work, solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) was developed for investigation of lung cancer volatile biomarkers. Headspace SPME conditions (fiber coating, extraction temperature and extraction time) and desorption conditions were optimized and applied to determination of volatiles in human blood. To find the biomarkers of lung cancer, investigation of volatile compounds in lung cancer blood and control was performed by using the present method. Concentrations of hexanal and heptanal in lung cancer blood were found to be much higher than those in control blood. The two molecules of hexanal and heptanal were regarded as biomarkers of lung cancer. By comparison of volatiles in breath and in blood, it is demonstrated that hexanal and heptanal in breath were originated from blood and screening of lung cancer by breath analysis be feasible. These results show that SPME/GC-MS is a simple, rapid and sensitive method very suitable for investigation of volatile disease markers in human blood.     

A computer-assisted study of the effect of gravitation on the functional state of the lungs

Gravitation influence on lung function was investigated in 115 patients and 21 healthy persons. Changing of an examinee's position from vertical into a horizontal one causes changes in lung function in health and disease. Characteristic features of lung function were determined for each group of examinees in order to improve the diagnosis and differential diagnosis of bronchial cancer and nonspecific chronic pulmonary diseases with a similar x-ray picture. A study of pulmonary ventilation and blood supply of the lungs in vertical and horizontal postures in Hodgkin's disease patients led to a conclusion that irradiation of patients seemed more effective in a phase of deep breath. It made it possible to reduce radiation exposure of healthy parts in a zone to be irradiated.     

Proteomic approaches to biomarker discovery in lung cancers by SELDI technology

Lung cancer is one of the most common tumors all over the world and one of those with higher mortality in clinic. For instance, 169500 new cases of lung cancer were estimated in the United States for 2001[1]. In recent years, both morbidity and mortality of lung cancer were reported gradually increasing in our country. Therefore, it has become an urgent task to search and discover specific biomarkers for lung cancer. In tumor genesis, certain cellular proteins must have changed their express…     

Proteomic approaches to biomarker discovery in lung cancers by SELDI technology

The purpose of the present work is to identify protein profiles that could be used to discover specific biomarkers in serum and discriminate lung cancer. Thirty serum samples from patients with lung cancer (15 cases of primary brochogenic carcinoma, 9 cases of metastasis lung cancer and 6 cases of lung cancer after chemotherapy) and twelve from healthy individuals were analyzed by SELDI (Surfaced Enhanced Laser Desorption/Ionization) technology. Anion-exchange columns were used to fractionate the sera with 6 designated pH washing solutions. Two types of protein chip arrays, IMAC-Cu and WCX2, were employed. Protein chips were examined in PBSII ProteinChip Reader (Ciphergen Biosystems Inc.) and the resulting profiles between cancer and normal were analyzed with Biomarker Wizard System. In total, 15 potential lung cancer biomarkers, of which 6 were up-regulated and 9 were down-regulated, were discovered in the serum samples from patients with lung cancer. 5 of 15 these biomarkers were able to be detected on both WCX2 and IMAC-Cu protein chips. The sensitivities provided by the individual markers range from 44.8% to 93.1% and the specificities were 85.0%–94.4%. Our results suggest that serum is a capable resource for detection of lung cancer with specific biomarkers. Moreover, protein chip array system was shown to be a useful tool for identification, as well as detection of disease biomarkers in sera.     

Effect of age on the profile of alkanes in normal human breath

Ethane and pentane in breath are markers of oxidative stress, produced by ROS-mediated lipid peroxidation of n-3 and n-6 polyunsaturated fatty acids (PUFAs), but little is known about other n-alkanes in normal human breath. We investigated the spectrum of alkanes in normal human alveolar breath, and their variation with age. Fifty normal humans were studied (age range 23-75, median 35). Volatile organic compounds (VOCs) in alveolar breath were captured on sorbent traps and assayed by gas chromatography and mass spectroscopy. Alveolar gradients (concentration in breath minus concentration in ambient room air) of alkanes were determined. C4-C20 alkanes were observed in breath and room air. Their mean alveolar gradients were negative from C4 to C12 and positive from C13 to C20. The mean alveolar gradients of four alkanes (C5-C8) were significantly less negative in the older subjects (p < 0.05). There were no significant differences between males and females. Normal human breath contained a spectrum of alkanes which may include new markers of oxidative stress. The mean rate of clearance (via cytochrome p450) exceeded the mean rate of synthesis (by ROS-mediated oxidative stress) for C4-C12 alkanes, while synthesis was greater than clearance for C13-C20 alkanes. The elevated alkane profile in older subjects was consistent with an age-related increase in oxidative stress, though an age-related decline in alkane clearance rate may have contributed.     

Hydrocarbon emulsification and enhanced crude oil degradation by lauroyl glucose ester

Enzymatically synthesized lauroyl glucose emulsified different hydrophobic substrates when assayed spectrophotometrically. Stable emulsions were formed with triglycerides as well as with hydrocarbons. There was a linear relation between the concentration of lauroyl glucose (50-450 microg) and emulsification activity under the assay conditions when tested with aromatic and aliphatic hydrocarbons. This sugar ester was able to emulsify the aromatic hydrocarbons benzene, toluene and xylene. Long chain alkanes (n-decane and n-hexadecane) as well as brominated long chain alkanes (1-bromodecane and 1-bromohexadecane) were efficiently emulsified. The effect of lauroyl glucose ester on degradation of crude oil by a known oil-degrading Rhodococcus species was also investigated. The culture showed enhanced degradation of crude oil when lauroyl glucose ester was used as an emulsifier. It degraded 70% of the aliphatic fraction of Bombay High crude oil in the presence of the sugar ester at a concentration of 200mg l(-1) as compared to 50% without the emulsifier.     

Differential MicroRNAs Expression in Serum of Patients with Lung Cancer, Pulmonary Tuberculosis, and Pneumonia

MicroRNAs (miRNAs) play critical regulatory roles in the physiological and pathological processes. The high stability of miRNAs in human serum represents attractive novel diagnostic biomarkers of clinical conditions. Several studies have shown that aberrant expression of miRNAs in human cancer including lung cancer, but little is known about their effects on some infectious lung diseases such as pulmonary tuberculosis (TB) and pneumonia. In this study, we investigated miRNA expression pattern in serum of Egyptian patients with lung cancer, TB, and pneumonia compared with matched healthy controls. Using microarray-based expression profiling followed by real-time quantitative polymerase chain reaction validation, we compared the levels of a series of circulating miRNAs (miR-21, miR-155, miR-182, and miR-197) in serum from patients with lung cancer (n = 65), pulmonary tuberculosis (n = 29), pneumonia (n = 29), and transudate (n = 16) compared with matched healthy controls (n = 37). MiRNA SNORD68 was the housekeeping endogenous control. We found that the serum levels of miR-21, miR-155, and miR-197 were significantly elevated in the patients with lung cancer and pneumonia whereas miR-182 and miR-197 levels were increased only in patients with lung cancer and TB, respectively, compared with controls. Receiver operating characteristic analysis revealed that miR-182, miR-155, and miR-197 have superior diagnostic potential in discriminating patients with lung cancer, pneumonia, and TB, respectively, from controls. Our results conclude that the differential expression of the four studied miRNAs can be potential non-invasive biomarkers for patients with lung cancer, TB and pneumonia.     

Application of gas chromatography mass spectrometry (GC–MS) in conjunction with multivariate classification for the diagnosis of gastrointestinal diseases

Gastrointestinal diseases such as irritable bowel syndrome, Crohn’s disease (CD) and ulcerative colitis are a growing concern in the developed world. Current techniques for diagnosis are often costly, time consuming, inefficient, of great discomfort to the patient, and offer poor sensitivities and specificities. This paper describes the development and evaluation of a new methodology for the non-invasive diagnosis of such diseases using a combination of gas chromatography mass spectrometry (GC–MS) and chemometrics. Several potential sample matrices were tested: blood, breath, faeces and urine. Faecal samples provided the only statistically significant results, providing discrimination between CD and healthy controls with an overall classification accuracy of 85 % (78 % specificity; 93 % sensitivity). Differentiating CD from other diseases proved more challenging, with overall classification accuracy dropping to 79 % (83 % specificity; 68 % sensitivity). This diagnostic performance compares well with the gold standard technique of colonoscopy, suggesting that GC–MS may have potential as a non-invasive screening tool.