Generation of volatile compounds on mouth exposure to urea and sucrose: implications for exhaled breath analysis

The increase in ammonia and ethanol in the exhaled breath stream following mouthwashes by aqueous solutions of urea and sugar (sucrose), respectively, has been investigated by analysing exhaled breath in real time using selected ion flow tube mass spectrometry, SIFT-MS. It is shown that the measured levels of these compounds in the stream of exhaled breath can be much greater than the endogenous levels originating at the alveolar boundary. Thus, it is concluded that without careful preparation, mouth production of these compounds, and other compounds as yet unidentified, can seriously compromise the quantification of truly endogenous trace compounds present in blood and in the alveolar breath, as required for clinical diagnosis, and can probably introduce additional compounds into the breath stream that could seriously mislead breath analysis. The concentrations of both the urea and sucrose solutions used to enhance the ammonia and ethanol levels were larger than normally present in food and drinks and so in most situations such severe enhancements will not occur.     

Update on urine as a biomarker in cancer: a necessary review of an old story

ABSTRACT

Introduction: Cancer causes thousands of deaths worldwide each year. Therefore, monitoring of health status and the early diagnosis of cancer using noninvasive assays, such as the analysis of molecular biomarkers in urine, is essential. However, effective biomarkers for early diagnosis of cancer have not been established in many types of cancer.

Areas covered: In this review, we discuss recent findings with regard to the use of urine composition as a biomarker in eleven types of cancer. We also highlight the use of urine biomarkers for improving early diagnosis.

Expert opinion: Urinary biomarkers have been applied for clinical application of early diagnosis. The main limitation is a lack of integrated approaches for identification of new biomarkers in most cancer. The utilization of urinary biomarker detection will be promoted by improved detection methods and new data from different types of cancers. With the development of precision medicine, urinary biomarkers will play an increasingly important clinical role. Future early diagnosis would benefit from changes in the utilization of urinary biomarkers.     

Enzymes and related proteins as cancer biomarkers: a proteomic approach

BACKGROUND: The discovery of cancer biomarkers has become a major focus of cancer research, which holds promising future for early detection, diagnosis, monitoring disease recurrence and therapeutic treatment efficacy to improve long-term survival of cancer patients. Most of the functional information of the cancer-associated genes resides in the proteome. Since cancer is a complex disease, it might require a panel of multiple biomarkers in order to achieve sufficient clinical efficacy. METHODS: Serum/plasma is the most accessible biological specimen collected from patients. Therefore, serum proteomic diagnostics would be the most promising new test for cancer. With the advent of new and improved proteomic technologies, such as protein chips and mass spectrometry coupled with advanced bioinformatic tools, it is possible to develop potential cancer biomarkers. However, specimen collection, handling, study design and data analysis are essential components for successful biomarker discovery and validation. Multi-center case control study should be conducted with extensive clinical validation to minimize the impact of possible confounding variables (non-biological). CONCLUSIONS: Enzymes and related proteins, such as inhibitors, are promising candidates for cancer diagnostics.     

An off-line breath sampling and analysis method suitable for large screening studies

We present a new, off-line breath collection and analysis method, suitable for large screening studies. The breath collection system is based on the guidelines of the American Thoracic Society for the sampling of exhaled NO. Breath containing volatile gases is collected in custom-made black-layered Tedlar bags and analyzed by proton-transfer reaction mass spectrometry (PTR-MS). The collection method and data analysis is validated for its accuracy, precision, selectivity, limits of detection, sensitivity and reproducibility. Consecutive fillings of five bags by the same person gave reproducible results to within 12% relative standard deviation (RSD) for methanol, acetaldehyde, acetone and water content from breath, whereas isoprene was constant to within 30% RSD. In an exploratory small-scale case-control study, we monitor the exhaled breath of 11 lung cancer patients on the day before surgery. The control group consisted of 57 age-matched subjects, the so-called 'healthy smokers'. This study is used as an example of the use of the system presented here.     

Lung cancer biomarkers in exhaled breath

Lung cancer is the leading cause of cancer-related mortality worldwide. Methods for early detection of lung cancer, such as computerized tomography scanning technology, often discover a large number of small lung nodules, posing a new problem to radiologists and chest physicians. The vast majority of these nodules will be benign, but there is currently no easy way to determine which nodules represent very early lung cancer. Adjuvant testing with PET imaging and nonsurgical biopsies has a low yield for these small indeterminate nodules, carries potential morbidity and is costly. Indeed, purely morphological criteria seem to be insufficient for distinguishing lung cancer from benign nodules at early stages with sufficient confidence, therefore false positives undergoing surgical resection frequently occur. A molecular approach to the diagnosis of lung cancer through the analysis of exhaled breath could greatly improve the specificity of imaging procedures. A biomarker-driven approach to signs or symptoms possibly due to lung cancer would represent a complementary tool aimed at ruling out (with known error probability) rather than diagnosing lung cancer. Volatile and nonvolatile components of the breath are being studied as biomarkers of lung cancer. Breath testing is noninvasive and potentially inexpensive. There is promise that an accurate lung cancer breath biomarker, capable of being applied clinically, will be developed in the near future. In this article, we summarize some of the rationale for breath biomarker development, review the published literature in this field and provide thoughts regarding future directions.     

Exhaled breath condensate: a new method for lung disease diagnosis.

Analysis of exhaled breath composition in lung disease patients can indirectly point to biochemical changes that occur in the fluid lining airway surfaces. The parameters of redox and acid-base changes, and of inflammatory changes relevant in the pathogenesis of most pulmonary diseases are currently most widely determined in exhaled breath condensate. The collection of exhaled breath condensate is a safe, non-invasive, easy and simple diagnostic procedure that is suitable for longitudinal studies and applicable in patients of all age groups, irrespective of the disease severity. In spite of many scientific studies involving lung disease patients, methodology for exhaled breath condensate collection and analysis has not yet been realized for daily utilization. Additional studies of the exact origin of condensate constituents and standardization of the overall analytical process, including collection, storage, analysis and result interpretation, are needed. Irrespective of these limitations, further investigation of this sample type is fully justified by the fact that classical specimens used in the management of pulmonary disease are either obtained by invasive procedures (e.g., induced sputum, biopsy, bronchoalveolar lavage) or cannot provide appropriate information (e.g., urine, serum). Analysis of exhaled breath condensate in the future might contribute significantly to our understanding of the physiological and pathophysiological processes in lungs, to early detection, diagnosis and follow up of disease progression, and to evaluation of therapeutic response.     

Breath biomarkers for lung cancer detection and assessment of smoking related effects — confounding variables,influence of normalization and statistical algorithms

BackgroundUp to now, none of the breath biomarkers or marker sets proposed for cancer recognition has reached clinical relevance. Possible reasons are the lack of standardized methods of sampling, analysis and data processing and effects of environmental contaminants.MethodsConcentration profiles of endogenous and exogenous breath markers were determined in exhaled breath of 31 lung cancer patients, 31 smokers and 31 healthy controls by means of SPME-GC-MS. Different correcting and normalization algorithms and a principal component analysis were applied to the data.ResultsDifferences of exhalation profiles in cancer and non-cancer patients did not persist if physiology and confounding variables were taken into account. Smoking history, inspired substance concentrations, age and gender were recognized as the most important confounding variables. Normalization onto PCO₂ or BSA or correction for inspired concentrations only partially solved the problem. In contrast, previous smoking behaviour could be recognized unequivocally.ConclusionExhaled substance concentrations may depend on a variety of parameters other than the disease under investigation. Normalization and correcting parameters have to be chosen with care as compensating effects may be different from one substance to the other. Only well-founded biomarker identification, normalization and data processing will provide clinically relevant information from breath analysis.     

Quantification of recent smoking behaviour using proton transfer reaction-mass spectrometry (PTR-MS)

Smoking is the most important single risk factor in current public health. Surveillance of exposure to tobacco smoke may be accomplished using environmental monitoring or in-vivo tests for smoking biomarkers. Acetonitrile exhaled in human breath has been described as a potential marker mirroring recent smoking behavior. The aim of this study was to determine exhaled acetonitrile levels in a sample of 268 volunteers (48 smokers, 220 non-smokers) attending a local health fair. Breath specimens were collected into inert sample bags, with parallel collection of ambient air. Subsequently, all samples were analysed using proton transfer reaction-mass spectrometry (PTR-MS). Smokers had elevated levels of exhaled acetonitrile compared with non-smokers (p<0.001). Analysis using the receiver-operating-characteristic curve demonstrated that smoking can be predicted with a sensitivity of 79% and a specificity of 91%, using a cut-off concentration of 20.31 parts per billion of acetonitrile. This first field survey of exhaled acetonitrile in a large group of test persons demonstrates the feasibility of a rapid and non-invasive test for recent exposure to tobacco. We conclude that analysis of exhaled-breath acetonitrile may serve as a method of determining recent active smoking behaviour.     

Mass spectrometry for malaria diagnosis

A physical method currently being developed for malaria parasite detection and diagnosis in blood is reviewed in this article. The method - direct laser desorption mass spectrometry - is based on the detection of heme (iron protoporphyrin) as a unique qualitative and quantitative molecular biomarker for malaria. In infected erythrocytes, the parasite sequesters heme in a molecular crystal (hemozoin) - a volume of highly concentrated and purified biomarker molecules. Laser desorption mass spectrometry detects only heme from hemozoin in parasite-infected blood, and not heme that is bound to hemoglobin or other proteins in uninfected blood samples. The method requires only a drop of blood with minimal sample preparation. Laser desorption mass spectrometry may become a rapid and high-throughput tool for specific and sensitive pan-malaria detection at levels below 10 parasites/mul of blood.     

SELDI proteinchip MS: a platform for biomarker discovery and cancer diagnosis

Identification and understanding the structures, interactions and functions of all of a cell's proteins is one of the major goals of the postgenome era. The genome project has produced a wealth of information that is greatly expounding the genetic basis of cancer. However, it falls short in not allowing for accurate prediction of what is happening at the protein level in a cancer cell or a body fluid proteome. It is the hope that, by deciphering the alterations in the cancer proteome, biomarkers and patterns of biomarkers will be found that will lead to improvements in early detection, diagnosis and treatment monitoring. To achieve this goal, rapid high-throughput proteomic technologies will be required. The SELDI ProteinChip Biomarker mass spectrometry system appears to have potential in this effort, both for biomarker discovery and as a potential clinical diagnostic assay platform.     

呼气冷凝液中3种肿瘤标志物联合检测在肺癌诊断中的价值研究

目的对呼气冷凝液（EBC）中3种肿瘤标志物进行联合检测,研究其在肺癌诊断中的临床价值,探讨该方法在临床应用的可能性,为今后的肺癌诊断和治疗效果评估提供可靠的参考依据。方法选择2011年1月15日至2012年12月15日该院肿瘤科收治的肺癌患者30例作为肺癌组,另抽取同期健康体检者30例作为健康对照组,对肺癌组患者治疗前后和健康对照组进行EBC及血清中癌胚抗原（CEA）、细胞角蛋白19的可溶性片段（CYFRA21—1）、血管内皮生长因子（VEGF）水平检测,并对比分析检测结果。采用标准EBC收集器收集EBC,CEA、CYFRA21—1采用化学发光法,VEGF用酶联免疫吸附法测定。结果肺癌组患者EBC及血清中CEA、CY—FRA21-1、VEGF检测水平明显高于对照组（P〈0．05）,化疗后CEA、CYFRA21-1、VEGF水平较治疗前显著降低（P〈O．05）,EBC中cEA、CYFRA21-1、VEGF水平较血清中低（P〈0．05）。结论对EBC中CEA、CYFRA21—1、vEGF水平进行检测对于肺癌的诊断、病理分型和疗效判断均具有重要的参考价值,EBC检测结果与血清结果联合可实现相互补充,提高阳性率,值得关注。     

Label-free mass spectrometry-based proteomics for biomarker discovery and validation

For most diseases, better biomarkers are urgently needed to enable (early) detection, diagnosis, prognosis, stratification for therapy and response monitoring. Proteomics delineates gene products that carry out the majority of cellular functions, and thereby may not only yield insight into altered signaling pathways in disease, but also yield novel biomarkers. In recent years, great progress has been made in mass spectrometry-based analysis of clinical tissues and biofluids, with identification and quantification of thousands of proteins now becoming increasingly routine. However, biomarker validation and clinical translation has turned out to be challenging. In this review, we summarize current mass spectrometry-based proteomics strategies for biomarker discovery and verification using selected reaction monitoring, with a focus on progress and recent applications in clinical material using label-free approaches.     

Advanced multimodal diagnostic approaches for detection of lung cancer

Introduction: Lung cancer (LC) emerges as a principle cause of death among smokers and is also one of the most lethal forms of cancer in nonsmokers. LC is mainly classified as non-small cell lung cancer (NSCLC), small cell LC, and lung carcinoid tumor. NSCLC is the most prevalent form of LC and its early stage diagnosis is essential to reduce mortality rate of patients and provide specific therapy. The field of LC diagnostics witnessed a gradual escalation with advancement in technology.

Areas covered: This comprehensive review focuses on classification of LC and advanced diagnostics for LC detection like biosensors, biomarkers, nanotechnology-based diagnostics, wearable devices, mobile health, artificial intelligence (AI), aptamers, and molecularly imprinted polymers (MIPs).

Expert opinion: Liquid biopsy and breath analysis developments are the most promising and advanced technologies for the detection of biomarkers associated with LC. Wearable devices and AI are two niche areas that require development and standardization for commercialization. The upcoming technology based on nanosystems includes robots, fibers, and particles for sensitive detection of LC. In the near future, nanotechnology-based theranostics, aptamers, and MIPs will emerge in early-stage diagnosis of LC.     

Potential of surface acoustic wave biosensors for early sepsis diagnosis

Early diagnosis of sepsis is a difficult problem for intensivists and new biomarkers for early diagnosis have been difficult to come by. Here we discuss the potential of adapting a technology from the electronics industry, surface acoustic wave (SAW) sensors, for diagnosis of multiple markers of sepsis in real time, using non-invasive assays of exhaled breath condensate. The principles and advantages of the SAW technology are reviewed as well as a proposed plan for adapting this flexible technology to early sepsis detection.     

Clinical proteomics: the promises and challenges of mass spectrometry-based biomarker discovery

The lack of effective surrogates or biomarkers for biologic and disease state is a major hindrance to effective cancer care. Many laboratories have been using mass spectrometry as the means to discover new biomarkers. The success of these efforts relies on effective collaboration between clinicians and mass spectroscopists. This review describes many of the common proteomic experimental methodologies and evaluates the current state of proteomics-based biomarker discovery--starting from sample collection through sample processing and ultimately sample measurement. Guidelines are given for the evaluation of experimental design and for the gauging of result significance. Particular emphasis is placed on those aspects strongly associated with reproducibility from both clinical and laboratory perspectives.     

Peptides in body fluids and tissues as markers of disease

The general awareness of the importance of peptides in physiology and pathophysiology has increased strongly over the last few years. With worldwide progress in the analysis of whole genomes, the knowledge base in gene sequence and expression data useful for protein and peptide analysis has drastically increased. The medical need for relevant biomarkers is enormous. This is particularly true for the many types of cancer, but other diseases such as Type 2 diabetes also lack useful and adequate diagnostic markers with high specificity and sensitivity. Despite advances in imaging technologies for early detection of diseases, proteomic and peptidomic multiplex techniques have evolved in recent years. This review focuses on the application of peptidomics technologies to peptides in health and disease. Peptidomics technologies provide new opportunities for the detection of low-molecular-weight proteome biomarkers (peptides) by mass spectrometry. Improvements in peptidomics research are based on separation of peptides and/or proteins by their physicochemical properties in combination with mass spectrometric detection, identification and sophisticated bioinformatics tools for data analysis. Therefore, peptidomics technologies offer an opportunity to discover novel biomarkers for diagnosis and management of disease (e.g., prognosis, treatment decision and monitoring response to therapy).     

Preanalytic influence of sample handling on SELDI-TOF serum protein profiles

BACKGROUND: High-throughput proteomic methods for disease biomarker discovery in human serum are promising, but concerns exist regarding reproducibility of results and variability introduced by sample handling. This study investigated the influence of different preanalytic handling methods on surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) protein profiles of prefractionated serum. We investigated whether older collections with longer sample transit times yield useful protein profiles, and sought to establish the most feasible collection methods for future clinical proteomic studies. METHODS: To examine the effect of tube type, clotting time, transport/incubation time, temperature, and storage method on protein profiles, we used 6 different handling methods to collect sera from 25 healthy volunteers. We used a high-throughput, prefractionation strategy to generate anion-exchange fractions and examined their protein profiles on CM10, IMAC30-Cu, and H50 arrays by using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. RESULTS: Prolonged transport and incubation at room temperature generated low mass peaks, resulting in distinctions among the protocols. The most and least stringent methods gave the lowest overall peak variances, indicating that proteolysis in the latter may have been nearly complete. For samples transported on ice there was little effect of clotting time, storage method, or transit time. Certain proteins (TTR, ApoCI, and transferrin) were unaffected by handling, but others (ITIH4 and hemoglobin beta) displayed significant variability. CONCLUSIONS: Changes in preanalytical handling variables affect profiles of serum proteins, including proposed disease biomarkers. Proteomic analysis of samples from serum banks collected using less stringent protocols is applicable if all samples are handled identically.     

High-resolution serum proteomic profiling of Alzheimer disease samples reveals disease-specific, carrier-protein-bound mass signatures

BACKGROUND: Researchers typically search for disease markers using a "targeted" approach in which a hypothesis about the disease mechanism is tested and experimental results either confirm or disprove the involvement of a particular gene or protein in the disease. Recently, there has been interest in developing disease diagnostics based on unbiased quantification of differences in global patterns of protein and peptide masses, typically in blood from individuals with and without disease. We combined a suite of methods and technologies, including novel sample preparation based on carrier-protein capture and biomarker enrichment, high-resolution mass spectrometry, a unique cohort of well-characterized persons with and without Alzheimer disease (AD), and powerful bioinformatic analysis, that add statistical and procedural robustness to biomarker discovery from blood. METHODS: Carrier-protein-bound peptides were isolated from serum samples by affinity chromatography, and peptide mass spectra were acquired by a matrix-assisted laser desorption/ionization (MALDI) orthogonal time-of-flight (O-TOF) mass spectrometer capable of collecting data over a broad mass range (100 to >300,000 Da) in a single acquisition. Discriminatory analysis of mass spectra was used to process and analyze the raw mass spectral data. RESULTS: Coupled with the biomarker enrichment protocol, the high-resolution MALDI O-TOF mass spectra provided informative, reproducible peptide signatures. The raw mass spectra were analyzed and used to build discriminant disease models that were challenged with blinded samples for classification. CONCLUSIONS: Carrier-protein enrichment of disease biomarkers coupled with high-resolution mass spectrometry and discriminant pattern analysis is a powerful technology for diagnostics and population screening. The mass fingerprint model successfully classified blinded AD patient and control samples with high sensitivity and specificity.     

Cancer diagnosis using proteomic patterns

The advent of proteomics has brought with it the hope of discovering novel biomarkers that can be used to diagnose diseases, predict susceptibility and monitor progression. Much of this effort has focused upon the mass spectral identification of the thousands of proteins that populate complex biosystems such as serum and tissues. A revolutionary approach in proteomic pattern analysis has emerged as an effective method for the early diagnosis of diseases such as ovarian cancer. Proteomic pattern analysis relies on the pattern of proteins observed and does not rely on the identification of a traceable biomarker. Hundreds of clinical samples per day can be analyzed utilizing this technology, which has the potential to be a novel, highly sensitive diagnostic tool for the early detection of cancer.