Exhaled particles and small airways.

BACKGROUND: Originally, studies on exhaled droplets explored properties of airborne transmission of infectious diseases. More recently, the interest focuses on properties of exhaled droplets as biomarkers, enabled by the development of technical equipment and methods for chemical analysis. Because exhaled droplets contain nonvolatile substances, particles is the physical designation. This review aims to outline the development in the area of exhaled particles, particularly regarding biomarkers and the connection with small airways, i e airways with an internal diameter < 2 mm. MAIN BODY: Generation mechanisms, sites of origin, number concentrations of exhaled particles and the content of nonvolatile substances are studied. Exhaled particles range in diameter from 0.01 and 1000 µm depending on generation mechanism and site of origin. Airway reopening is one scientifically substantiated particle generation mechanism. During deep expirations, small airways close and the reopening process produces minute particles. When exhaled, these particles have a diameter of < 4 µm. A size discriminating sampling of particles < 4 µm and determination of the size distribution, allows exhaled particle mass to be estimated. The median mass is represented by particles in the size range of 0.7 to 1.0 µm. Half an hour of repeated deep expirations result in samples in the order of nanogram to microgram. The source of these samples is the respiratory tract ling fluid of small airways and consists of lipids and proteins, similarly to surfactant. Early clinical studies of e g chronic obstructive pulmonary disease and asthma, reported altered particle formation and particle composition. CONCLUSION: The physical properties and content of exhaled particles generated by the airway reopening mechanism offers an exciting noninvasive way to obtain samples from the respiratory tract lining fluid of small airways. The biomarker potential is only at the beginning to be explored.

Increased nitric oxide in exhaled air after intake of a nitrate-rich meal.

Exhaled and nasal NO (ENO, NNO) have been suggested as markers for inflammation in lower and upper respiratory tract respectively. It is still unknown how a number of factors, apart from airway inflammation, can influence NO levels. The aim of this study was to determine the effect of a nitrate-rich meal on ENO and NNO. Sixteen healthy subjects were observed during 1 week on normal diet before a nitrate-restricted diet was introduced in the next. On day 3 of the second week they were made to ingest a nitrate rich meal. ENO, NNO, plasma nitrate and plasma L-arginine were followed before the meal and afterwards for 3 h. ENO and NNO as well as plasma nitrate and plasma L-arginine were significantly elevated after the nitrate-rich meal. The median maximal increase of ENO and NNO was 47% and 13% respectively. We found a moderate but significant correlation between the rise in plasma nitrate and ENO (r(s)=0.57, P=0.027) but none between plasma nitrate and NNO (r(s)=-0.02, P=0.95). As nitrate in the diet seems to substantially influence the levels of ENO it is important either to restrict or register the intake of nitrate-rich food prior to measuring ENO.

Nitric oxide (NO) in exhaled air after experimental ozone exposure in humans.

We hypothesized that ozone, a common air pollutant, potent in producing airway inflammation, would increase the production of exhaled nitric oxide (NO). If so, measurement of exhaled NO could potentially be a valuable tool in population studies of air pollution effects. Eleven healthy non-smoking volunteers were exposed to 0.2 ppm ozone (O3) and filtered air for 2h on two separate occasions. Exhaled NO and nasal NO were measured before and on five occasions following the exposures. Changes in exhaled and nasal NO after ozone exposure were adjusted for changes after air exposure. There was a slight decrease in exhaled NO (-0.6; -3.1-1.2 ppb) (median and 95% confidence interval) and of nasal NO (-57; -173-75 ppb) directly after the ozone exposure. No significant changes in exhaled or nasal NO were however found 6 or 24 h after the exposure. Within the examined group, an O3 exposure level proven to induce an airway inflammation caused no significant changes in exhaled or nasal NO levels. Hence, the current study did not yield support for exhaled NO as a useful marker of ozone-induced oxidative stress and airway inflammation after a single exposure. This contrasts with data for workers exposed to repeated high peaks of ozone. The potential for exhaled NO as a marker of oxidative stress therefore deserves to be further elucidated.

Nasal nitric oxide and its relationship to nasal symptoms, smoking and nasal nitrate.

Nitric oxide (NO) is produced in the nasal mucosa and in the paranasal sinuses. Increased nasal NO concentrations have been found in patients with asthma and/or rhinitis, and nasal NO has been suggested to be a marker of nasal inflammation. Measuring the stable end products of NO, nitrate and nitrite in nasal lavage fluid have been proposed as an indirect method for measuring NO concentration. The aim of this study was to measure nasal NO concentration, and to find out its relationship to nasal nitrate concentration and clinical parameters. 73 paper-mill workers were investigated with nasal and exhaled NO, nitrate in nasal lavage fluid and were given a respiratory questionnaire. Nasal air was sampled directly from a nasal mask and NO concentration was measured with a chemiluminescence analyser. Exhaled NO was measured with the subjects breathing tidal volumes and wearing nose clips. The nitric oxide metabolites were analysed as nitrate, after reduction of nitrite to nitrate. Smokers had lower nasal NO concentration (264 ppb) as compared to NO concentrations of 340 ppb among non-smokers (p = 0.02). There was no statistically significant relationship between nasal NO concentration and nitrate in nasal lavage fluid or nasal symptoms. Nasal NO concentration was significantly related to FVC (p = 0.047) and there was a relationship with borderline statistical significance (p = 0.06) to FEV1. In conclusion, we found no relationship between nitrate in nasal lavage and nasal NO, and neither of these were correlated to nasal symptoms or to nasal PIF. Nasal NO was significantly lower among smokers. Further controlled studies on subjects with rhinitis are needed, to evaluate the relation between nasal NO and nasal inflammation. In addition, there is also a need to develop methods for measuring nasal NO that minimise contamination from sinuses.

A field method for sampling benzene in end-exhaled air.

A simple and reliable field method is presented for sampling and analysis of benzene in end-exhaled air. The sample is collected directly on an adsorbent tube while the subject exhales through a sampling device consisting of a modified peak expiratory flow meter. To ensure sampling of end-exhaled air, the temperature of the breath is monitored during expiration. The analytes subsequently are thermally desorbed and analyzed by gas chromatography. No sample preparation before analysis is needed, and therefore sample loss is minimized, shipping is easy, storage is possible, and clean up is unnecessary. All these steps have been major problems in earlier methods for breath analysis. The presented method has been applied to the monitoring of benzene. The separation of benzene from other components of exhaled air was good and the detection limit low (0.5 microgram/m3), and therefore benzene could be monitored in occupationally nonexposed nonsmokers. No carry-over in the sampling device or breakthrough could be detected. The samples were stable for at least a week. The combined precision in sampling and analysis was excellent, with a coefficient of variation of 13%.

Determination of low concentrations of benzene in urine using multi-dimensional gas chromatography.

A method for the determination of benzene in urine of occupationally or environmentally exposed persons was developed. The method was based on dynamic headspace, preconcentration on a solid sorbent, followed by thermal desorption and gas chromatographic determination. To achieve sufficient selectivity, we used multi-dimensional gas chromatography in combination with the inexpensive and robust flame ionisation detector. The limit of detection was 7 ng l-1 and the limit of quantification was 23 ng l-1. The linearity was good (correlation coefficient 0.999) in the range examined (20-4000 ng l-1) and the repeatability was 9%. The average recovery at low concentrations (20-400 ng l-1) was 86%. Analysis of a certified reference material of benzene in water, traceable to NIST, did not differ significantly from the certified value. Samples, frozen (-20 degrees C) in glass bottles sealed with Teflon-silicon septa, were stable for 1 year and refrigerated samples (4 degrees C) for at least 1 week. Loss of benzene during the collection and transfer of urine was investigated and found to be acceptable. The method is a cost effective and robust alternative to GC-MS and permits reliable quantification of occupational exposure and, in most cases, also of urine concentrations that can be expected from environmental exposure.

Specific determination of benzene in urine using dynamic headspace and mass-selective detection.

A method for the determination of benzene in urine was developed, based on dynamic headspace and preconcentration of the analyte on a solid sorbent. The subsequent analysis by thermal desorption of the sorbent, capillary gas chromatography and mass-selective detection ascertained a low limit of detection (6.5 ng/l) and a highly specific determination. The limit of detection is an order of magnitude lower than that reported earlier and allows reliable quantitation of occupational exposure and of most environmental exposures. Samples could be stored frozen for at least a month without significant loss.

Cardiac arrhythmia in refrigerator repairmen exposed to fluorocarbons.

A field study of 89 refrigerator repairmen was carried out to ascertain whether occupational exposure to fluorocarbons induces cardiac arrhythmia. The concentrations of fluorocarbons in the breathing zones and the heart activity were recorded simultaneously. Most cooling systems contained FC 12 or FC 22. The highest level recorded in one minute was 14,000 ppm and the highest time weighted level during eight hours was 280 ppm. Two types of arrhythmia were recorded, ectopic beats and sudden bradycardia. A within subject comparison design was applied and the main parameter was the difference in arrhythmia frequencies between exposed and unexposed periods. No appreciable differences between exposed and unexposed periods and no consistent dose effect relations were observed, although subjects in the medium exposure category showed a difference of borderline significance (Wilcoxon's test: p = 0.05, one tailed). The frequencies of arrhythmia when unexposed were somewhat higher than previously reported. Misclassification of the exposure and the possible confounding effect of physical workload and psychological strain may have obscured a causal relation and therefore a minor effect cannot be ruled out. The results do not support the notion that fluorocarbons induce cardiac arrhythmia in occupationally exposed refrigerator repairmen.

Exhaled nitric oxide among pulpmill workers reporting gassing incidents involving ozone and chlorine dioxide.

The aim of the study was to investigate whether measurement of nitric oxide in exhaled air could be used for assessing the effects of irritants on the respiratory system, in this case recurrent ozone gassing in an occupational setting. The study population comprised bleachery workers (n=56) from a Swedish pulpmill carrying out ozone-based pulp bleaching since 1992 and controls (n=39). Both groups were investigated by measuring NO in exhaled air, methacholine challenge test and answers to a questionnaire concerning history of respiratory symptoms and accidental exposure to ozone peaks. There was no significant difference in NO output between exposed subjects and controls (median 67.2 versus 55.0 nL x min(-1), p=0.64). However, among bleachery workers reporting ozone gassings, the median NO output was 90.0 nL x min(-1) compared to 58.8 nL x min(-1) among those not reporting such incidents (p=0.019). There was no relation between exhaled NO and the prevalence of respiratory symptoms or bronchial hyperresponsiveness. In a multiple regression model, only reported ozone gassings were associated (p=0.016) with NO output. The results indicate an association between previous response to ozone gassing and nitric oxide output. The increased nitric oxide output among the bleachery workers reporting peak ozone exposure may indicate that chronic airway inflammation is present. Further studies are needed to evaluate the extent to which nitric oxide can be used for biological monitoring of respiratory health effects, and to relate it to other markers of airway inflammation.

Acetic aldehyde and formaldehyde in cutting fluids and their relation to irritant symptoms.

The objective was to study the formation of acetic aldehyde in cutting fluids and its relation to irritation of mucous membranes and the skin. Acetic aldehyde and formaldehyde were analysed in two large cutting fluid systems in an engineering industry. Samples were taken 1-5 times a week during a year. Concentration of the cutting fluid, leakage oils, pH, bacteria, yeast and fungi were analysed weekly. The occurrence of mucous membrane irritation was registered through questionnaires to the exposed workers. About 50 persons were exposed to each of the cutting fluids. The concentration of the aldehydes varied with time and between the cutting fluids. None of the analysed parameters could explain the variable concentration of aldehydes. Mucous membrane irritation was much more common in one of the systems, e.g. the prevalence of irritation in the nose was about 30-40% in workers exposed to a cutting fluid, while the corresponding prevalence was less than 10% in workers exposed to another fluid. The occurrence of symptoms was slightly associated with the concentration of aldehydes and pH of the fluid varied more in the fluid that caused most symptoms. A few measurements of ammonia indicated a higher concentration of ammonia in the fluid that caused most symptoms. It is concluded that irritation of mucous membranes and the skin may vary considerably between different cutting fluids of similar composition and use but the causal factor could not be determined in this study, but a variable pH and an increased concentration of ammonia may be indicators in this context. The concentration of acetic aldehyde vary with time and between cutting fluids with similar composition. A high variability may be an indicator of less stable cutting fluids. Better markers for the surveillance of cutting fluids needs to be developed as well as a health control programme.