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。     

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.     

Oxidative Stress in COPD and Its Measurement through Exhaled Breath Condensate

Oxidative stress and airway inflammation together form a vicious cycle, which is responsible for the disease progression in chronic pulmonary obstructive disease (COPD). The damaging effects of oxidative stress accumulate over the years, causing increased bronchial hyperresponsiveness and inflammation and destruction of airway epithelial cells and impairing the functions of antiproteases and surfactant. Although the lung expresses a number of antioxidants, cigarette smoking and recurrent infections associated with this disease overwhelm this protective mechanism. Studies of antioxidants in COPD have yielded conflicting results, probably due to the compartmentalization of these mediators, and because of the fact that the lung is a difficult organ to sample. Chronic exposure to oxidants upregulates the production of antioxidants, which become depleted during acute exacerbations. Future studies of the pathogenesis of COPD require a noninvasive yet accurate sampling procedure, of which exhaled breath condensate (EBC) is a good candidate. EBC samples the epithelial lining fluid, which contains the local oxidative stress markers in the lung. Oxidative stress markers such as hydrogen ions, hydrogen peroxide, 8‐isoprostanes, thiobarbituric acid reactive products, nitrosothiols, and nitrite/nitrate have been identified in EBC of COPD patients, whereas many other markers of the oxidative‐antioxidative balance have yet to be investigated.     

Exhaled breath analysis for early cancer detection: principle and progress in direct mass spectrometry techniques

Abstract Volatile biomarker analysis in exhaled breath is becoming one of the desirable strategies for cancer detection because it may offer a relatively inexpensive, rapid, and non-invasive screening method for early diagnosis. Breath analysis has attracted a considerable amount of scientific and clinical interest over the past decade. However, breath is not yet used for routine medical diagnostic purposes. Challenges faced in the development of breath analysis for cancer diagnosis include developing techniques that can measure biomarkers in exhaled breath at ultratrace levels, providing definitive evidence for their presence and for the relationship between the proposed biomarker and the underlying condition. Various analytical methods are used for the detection of breath biomarkers. Gas chromatography-based methods which involve sample collection, analyte preconcentration, desorption, and separation steps are the most popular. However, direct-sampling mass spectrometry techniques have been proven more reliable for air analysis without prior sample pretreatment or chromatographic separation. This review focuses on the most commonly used direct mass spectrometry methods for the direct online analysis of endogenous cancer biomarkers in exhaled breath, with particular attention to principle of detection, method performance, advantages, shortcomings, recent advances, and applications within health-related studies for cancer biomarkers research. The principle behind the science of breath analysis for cancer diagnosis is also discussed.     

慢性阻塞性肺疾病患者呼出气冷凝液中IL-8与8-异向前列腺素的变化及临床意义

目的观察慢性阻塞性肺疾病（COPD）患者呼出气冷凝液（EBC）中IL-8与8-异向前列腺素水平的变化。方法对已确诊为COPD的30例患者（急性加重期和稳定期各15例）及15例健康志愿者（对照组）,采用呼出气冷凝液（EBC）收集器收集患者和健康志愿者的EBC,EBC中IL-8的检测采用酶联免疫吸附法（EHSA）、8-异向前腺素的检测采用酶免疫法（EIA）。同时测定肺功能。结果COPD急性加重期患者EBC中II,-8的水平为（861．1463±679．4282）ng·L-1,明显高于COPD稳定期患者的水平（416．2132±383．9651）ng·L-1（P〈O．05）;COPD急性加重期患者EBC中8-异向前列腺素的水平为（6．110±2．325）ng·L-1,明显高于COPD稳定期患者的水平（2．980±1．893）ng·L-1（P〈0．05）。COPD患者EBC中IL-8与FEVl％、FEVI±VC呈正相关（卸．396、0．512,均P〈0．01）;8一异向前列腺素与FEVl％、FEVlWVC呈负相关（r=-0．39、-0．76,均P〉0．05）。结论EBC的收集过程无创、安全、简便、可行。COPD患者EBC中的IL一8和8一异向前列腺素可作为评价COPD气道炎症反应、氧化应激状态和病情严重程度的较好指标。     

A noninvasive method to collect nasally exhaled air condensate in humans of all ages

BACKGROUND: The analysis of exhaled breath condensate may provide valuable insights into inflammatory and other metabolic processes of the lungs. However, its collection by active exhalation with conventional methods is cumbersome, demands a substantial level of co-operation with high motivation and is very difficult or impossible in children younger than about 4-6 years or in the elderly. A comfortable, noninvasive and efficient method is desirable. DESIGN AND PATIENTS: For collection a high-performance pump connected to a cold trap and nasal prongs were used. The volume of the condensate collected was assessed in 141 children of all ages and five adults. As an example for a low molecular component, H2O2 a marker of oxidative stress, was determined fluorometrically. RESULTS: On average, in healthy children from 4 weeks to 18 years of age, 84.0 (79.4, 87.3) microL min(-1) of nasally exhaled air condensate were collected. The volume obtained was about 45% less in 1-6-year-old children, increased linearly with collection time, and averaged about 20-30% of the exhaled water vapour. The concentration of H2O2 in the healthy children was 0.49 (0.48, 0.61) microM and did not depend on age, the time of the day, family, or personal history of atopy and sex. CONCLUSIONS: The method described is generally applicable, comfortable, noninvasive, safe and efficient and allows the collection of nasally exhaled air condensate for the evaluation of metabolic processes of the lungs.     

Effects of salbutamol on exhaled breath condensate biomarkers in acute lung injury: prospective analysis

Introduction  

The benefits of β-adrenergic stimulation have been described in acute lung injury (ALI), but there is still no evidence of its anti-inflammatory effect in these patients. Biomarkers in exhaled breath condensate (EBC) were used to study the effects of salbutamol on lung inflammation in mechanically ventilated patients with ALI.     

Volatile organic compounds in exhaled air from patients with lung cancer

Using a specially developed breath collection technique and computer-assisted gas chromatography/mass spectrometry (GC/MS), we have identified in the exhaled air of lung cancer patients several volatile organic compounds that appear to be associated with the disease. The GC/MS profiles of 12 samples from lung cancer patients and 17 control samples were analyzed by using general computerized statistical procedures to distinguish lung cancer patients from controls. The selected volatile compounds had sufficient diagnostic power in the GC/MS profiles to allow almost complete differentiation between the two groups in a limited patient population.     

Assessing airway inflammation: from guessing to quantitative measurements

Diagnosing asthma and following the response to treatment have relied on lung function measurements. Improved knowledge of the cellular events leading to airflow limitation has led to new clinical methods to assess the inflammatory component of the disease. Induced sputum analysis and exhaled nitric oxide (NO) measurements are already tools for clinical practice. New cell-specific inflammatory markers and further innovations in the testing of exhaled air, e.g. breath condensates and technical development of simple methods and devices, will also benefit the busy practitioner in near future. Assessing airway inflammation by quantitative measurements, instead of guessing, will also strengthen the role of anti-inflammatory medication as first-line treatment of asthma, even in its mildest forms.     

Assessing airway inflammation: from guessing to quantitative measurements

Diagnosing asthma and following the response to treatment have relied on lung function measurements. Improved knowledge of the cellular events leading to airflow limitation has led to new clinical methods to assess the inflammatory component of the disease. Induced sputum analysis and exhaled nitric oxide (NO) measurements are already tools for clinical practice. New cell-specific inflammatory markers and further innovations in the testing of exhaled air, e.g. breath condensates and technical development of simple methods and devices, will also benefit the busy practitioner in near future. Assessing airway inflammation by quantitative measurements, instead of guessing, will also strengthen the role of anti-inflammatory medication as first-line treatment of asthma, even in its mildest forms.     

Instrumentation for estimating pulmonary function in patients on positive end-expiratory pressure

For patients requiring PEEP during ventilation, e.g., patients with respiratory distress syndrome, performing traditional tests of lung function is a complicated problem. The pulmonary mechanics of these patients can change rapidly with potential for a negative impact on oxygenation. In an effort to address these problems, we designed a system with a three-way valve that permits the patient to be switched from the ventilator to a data collection system for the assessment of changes in the disease state. The short periods required for data collection (20 to 30 sec) do not interfere with patient care and are well within the limits of safety. A unique feature of the valved system is that it serves to maintain positive airway pressure during data collection. Tests show that, within the frequency range of interest, the mechanics of the measurement system are not altered by the DC pressures applied to the speaker and the valve itself does not contribute significantly to the serial impedance of the system.     

Metabolic disorders in patients with chronic obstructive pulmonary disease

AIM: To characterize cell dysregulation in chronic obstructive pulmonary disease (COPD) at local and systemic levels. MATERIAL AND METHODS: A total of 48 patients with COPD were examined (mean disease duration 11.2 +/- 7.2 years, mean age 58 +/- 4.2 years). Content of glutathione, and activity of glutathione peroxidase, glutathione reductase in whole blood (Novgorodtseva T.P., 2003), catalase in erythrocytes (Karpichenko A.I., 1999) were determined. Products of lipid peroxidation in exhaled breath condensate were defined to reveal diene conjugates of hydroperoxides, ketodienes, coupled trienes (Khyshiktuyev B.S., 1996). RESULTS: An evident oxidation stress at the local level was revealed in patients with COPD in deficiency of enzymatic antiperoxide link of anti-oxidant defense that is characterized by increased quantity of lipid peroxidation intermediants both in heptane and isopropanol phases.     

Nitrite in exhaled breath condensate as a marker of nitrossative stress in the airways of patients with asthma,COPD, and idiopathic pulmonary fibrosis

Background: Nitrite and nitrate are exhaled in droplets of an aerosol during breathing and can be assayed in the exhaled breath condensate (EBC) as markers of nitrossative stress in the airways of patients with asthma, COPD, and idiopathic pulmonary fibrosis (IPF). Subjects and methods: Using HPLC with fluorescence detection, nitrite and nitrate were assayed in EBC of 14 atopic patients with mild‐to‐moderate stable asthma, 18 atopic asthmatics with exacerbation, 14 COPD patients without exacerbation, 18 patients with exacerbated COPD, 13 patients with active IPF, and in 29 healthy subjects. Results: The geometric mean [exp(mean±SD)] EBC concentrations of nitrite (micromol/l) in patients with asthma [5.1(2.1–12.3)], exacerbation of asthma [5.1(2.8–9.6)], exacerbation of COPD [5.3(3.2–8.7)], and with IPF [5.5(2.9–10.2)] were higher (P<0.05) compared with those of healthy subjects [2.9(1.6–5.3)] and patients with stable COPD [3.0(1.3–6.7)]. Nitrite concentration increased with decreased lung function of patients with asthma (r_s=?0.31, P<0.02). Presumably owing to the contamination of the EBC sample with nitrate during collection, nitrate levels were highly variable among healthy subjects and higher compared with all groups of patients. Conclusion: EBC nitrite is a suitable marker of nitrossative stress in adult patients with lung diseases but cannot differentiate controlled and exacerbated asthma. Further improvements to the methods of EBC collection and sample handling are warranted. J. Clin. Lab. Anal. 24:317–322, 2010. © 2010 Wiley‐Liss, Inc.     

Exhaled breath condensate analysis in patients with COPD

Exhaled breath condensate (EBC) is a non-invasive method for studying the composition of airway lining fluid. EBC is mainly formed by water vapor but also contains aerosol particles in which several biomolecules including hydrogen peroxide, leukotrienes, prostaglandins, isoprostanes, nitric oxide-derived products, and hydrogen ions have been measured in healthy subjects. Some inflammatory mediators are elevated in patients with chronic obstructive pulmonary disease (COPD). Analysis of EBC has several advantages over other methods for assessing lung inflammation: it is completely non-invasive; this technique is particularly suitable for longitudinal studies; this method is potentially useful for assessing the efficacy of pharmacological therapy. Identification of selective profiles of inflammatory markers in EBC might also be of diagnostic value in patients with COPD. EBC analysis is currently more reliable for relative measures than for determining absolute levels of inflammatory mediators. The lack of standardization of the EBC analysis is currently the primary limitation of this technique making it difficult comparisons of data obtained in different laboratories. Reference analytical techniques are required to provide definitive evidence for the presence of several biomolecules in EBC and an accurate assessment of their concentrations in this biological fluid. Moreover, several methodological issues need to be addressed before this technique can be considered in the clinical management of patients with COPD. Despite important current limitations, further research in this area is warranted due to the lack of non-invasive methods for assessing lung inflammation which has a central role in the pathophysiology of COPD.     

非小细胞肺癌患者呼出气冷凝液VEGF检测的临床意义

目的测定非小细胞肺癌患者(NSCLC)呼出气冷凝液(EBC)中血管内皮生长因子(VEGF)并研究其临床意义。方法收集40例经病理确诊的NSCLC患者、20例稳定期的慢性阻塞性肺疾病(COPD)患者和20例健康体检者的EBC和血清标本,采用酶联免疫吸附法(ELISA)分别检测EBC和血清中VEGF水平。结果①NSCLC组EBC和血清中VEGF水平[(11.7±6.1)pg/mL、(310.5±23.9)pg/mL]显著高于COPD组[(6.3±4.9)pg/mL、(217.1±14.7)pg/mL]及正常对照组[(3.6±1.2)pg/mL、(162.5±16.5)pg/mL]。②EBC和血清中VEGF水平与NSCLC患者的TNM分期及淋巴结转移呈正相关,与肺癌病理类型及患者性别、年龄、吸烟史无密切关系。结论检测EBC中VEGF有助于肺癌的早期诊断、病情进展及预后判断。     

The gas chromatographic analysis of the fatty acids in the expired air in ischemic heart dis

Lipids of exhaled air condensate (EAC) and sweat are analyzed by gas chromatography in coronary patients with myocardial infarction complicated by lung edema. Changes of lipid fatty acid spectrum in EAC and sweat were uniform: the levels of linoleic and arachidonic acids were increased, which can aggravate the disease and lead to complications.     

Indices of lipid peroxidation in the condensate of exhaled air--a simple and objective method of estimating injuries to the pulmonary surfactant system]

A method for assessing injury to the pulmonary surfactant system is suggested, based on studies of lipid peroxidation parameters in exhaled air condensate. Free-radical processes in pulmonary surfactants were examined in normal subjects and in patients with various forms of chronic nonspecific diseases of the respiratory organs. The findings evidence that a disease essentially intensifies lipid peroxidation in the alveolar lining, this being parallelled by reduced antiradical defense. Such an approach to studies of pulmonary surfactant system injuries is marked for its simplicity and atraumatism, this recommending it for mass clinical check-ups. Use of this method is promising as regards the disease prognosis and assessment of the treatment efficacy, as well as detection of subjects at high risk of developing bronchopulmonary diseases.     

Breath analysis in critically ill patients: potential and limitations

Breath tests are attractive since they are noninvasive and can be repeated frequently in the dynamically changing state of critically ill patients. Volatile organic compounds can be produced anywhere in the body and are transported via the bloodstream and exhaled through the lung. They can reflect physiologic or pathologic biochemical processes such as lipid peroxidation, liver disease, renal failure, allograft rejection, and dextrose or cholesterol metabolism. This review describes the diagnostic potential of endogenous organic volatile substances in the breath of critically ill patients. Since many of these patients require ventilatory support, aspects of breath analysis under mechanical ventilation will be addressed. Analytical procedures, problems concerning the physiologic meaning of breath markers and future developments will be discussed.