The aim of the present study is to evaluate the effect of low (6 ml/kg) and high (12 ml/kg) tidal volume ventilation on inflammatory and oxidative stress biomarkers in the exhaled breath condensate (EBC) of ICU patients without lung injury. As the analysis of EBC is reflecting the composition of epithelial lining fluid (ELF), the study of EBC pH and inflammatory and oxidative stress markers could have the potential for assessing lung inflammation caused by mechanical ventilation. This study also aims to look at the possibility to identify selective profiles of biomarkers that might have a prognostic and/or diagnostic value in the follow up of these patients.

INTRODUCTION:

Airway inflammation plays a key role in many airway diseases. Non invasive methods to study these inflammatory processes and to monitor airway diseases are in high demand. Stimulating interest is in breath analysis, such as the analysis of exhaled breath condensate (EBC), a technique of sampling the epithelial lining fluid of the lung (ELF), as an even easier way to assess airway inflammation (1,2). The appeal of EBC lies in its ability to collect non-invasively a wide range of nonvolatile molecules from the respiratory tract, the fact that it can be repeated frequently within short intervals without adverse events and that collection devices can be used in a wide range of settings including intensive care units (3,4,5,6).

Analysis of EBC could not be limited to patient monitoring and understanding mechanisms of pulmonary disease. It also could become a useful tool for monitoring and screening for healthy individuals for possible early pulmonary tissue damage (6,7). There is a special need for more data on intra-subject and day-to-day variation, both essential for the decision as to whether a biomarker can serve as a research tool or even has the potential for disease monitoring in clinical practice (7).

It has been recognized for some time that mechanical ventilation may induce lung injury and inflammation (8,9,10). Recent experimental and clinical data suggest that in healthy lungs, mechanical ventilation with tidal volume ranging between 7 and 12 ml/kg in the absence of positive end-expiratory pressure may lead to endothelial, extracellular matrix and peripheral airways damage without major inflammatory response, which further worsen with higher tidal volumes (15,34). Several mechanisms may explain damage to the lung structure induced by mechanical ventilation: regional over distension, 'low lung volume' associated with tidal airway closure, and inactivation of surfactant (15).

High tidal volume ventilation has been shown to result in increased mortality while low tidal volume ventilation is regarded as a lung protective strategy in ALI, ARDS (11,12,13,35).

In contrast, in other randomized studies (31,32) including a heterogeneous group of major thoracic and abdominal surgical procedures, protective mechanical ventilation was not associated with a decrease in intrapulmonary and systemic inflammation. Furthermore, there was no evidence that protective ventilation prevented lung adverse effect or decreased systemic cytokine levels in cardiac surgery (33).

In line with these observations and considering that a practical parameter of increased mechanical stress of the lung remains to be demonstrated, studies may address the question whether the analysis of EBC biomarkers are related to ventilator-induced lung injury by low or high tidal volumes.

MATERIALS AND METHODS:

The present study is a prospective, randomized, controlled trial that will take place in the ICU of the University Hospital of Larissa, Thessaly. Authorization has been given from the Scientific Council and the ethical committee of our hospital.

Patients:

ICU patients requiring mechanical ventilation because of stroke, subarachnoid and/or intracerebral hemorrhage and with healthy respiratory system (evaluated using criteria as the LISS - Murray Lung Injury Severity Score) (14).

Interventions:

EBC collection will be performed on mechanically ventilated patients through the endotracheal tube according to ATS/ERS task force 2005 (7). Patients must be hemodynamically and respiratory stable.

All patients will be under sedation and will receive ventilation by volume control. The respiratory frequency will be adapted at the set tidal volume in order to maintain the pH values within normal limits (7.35-7.45). SaO2 will be maintained equal or superior to 95%.

EBC will be collected by inserting a special conduit (FILT, lung and chest diagnostics Ltd. Berlin Germany) for the breath condensate collecting device (Ecoscreen, Jaeger, Germany) into the expiratory limb of the ventilator tubing. Collecting time for EBC will be 30 min. No humidification will be used during the collection.

The acidity (pH) of EBC before and after de-aeration with an inert gas Argon, 350 ml/minutes for 10 minutes, (gas standardisation) (17) will be readily measured using a pH meter Jenway Model 3510.

All samples will be stored at -80 ο C for subsequent mediator measurements. Variables of ventilation (frequency, PEEP, FIO2, Vt), lung mechanics, arterial pressure, heart rate, arterial blood saturation, ICP and gas blood samples examination will be registered before and during the EBC collection. Also will be registered indices of lung injury (PiO2/FiO2, LISS), indices of systemic inflammation (temperature, leucocyte and neutrophil counts in blood samples) during the observation period. Disease severity indices (SOFA, SAPS, APACHE II) will be registered during the initial assessment.

EBC analysis:

EBC collected will be analyzed for pH, 8-isoprostane, H2O2, nitrites/nitrates and cytokines. The measurement of biomarkers will be performed using standardized procedures, as previously described.

pH measurements: pH will be measured as previously described (16,17). H2O2 measurements: H2O2 concentration will be determined by an enzymatic assay using horseradish peroxidase (Sigma Chemicals, St. Louis, MO), as previously described (17,18,19,20).

8-Isoprostane measurements: 8-Isoprostane will be determined by a competitive enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI), as previously described (17,18,21,29). The detection limit of the assay is 4 pg/ml.

Nitrogen oxides, nitrite/nitrate (NO2/NO3), and related products measurements: will be performed as previously described (17,22). Briefly, will be determined by using spectrophotometric assays (Griess reaction) (23,24,25,26,27,28).

Cytokines measurement: will be quantified by EIA/ELISA kits as previously described (24,25,29,30).

Protocol Details:

The patients after the initial assessment will be randomized to receive mechanical ventilation with 6 or 12 ml/Kg of ideal body weight calculated through the following equation:

For men [(Height (cm)-154) x 0.9] +50 and For women [(Height (cm)-154) x 0.9] +45.5. The observation period will be a minimum of 10 days (if possible) and EBC collection will be performed within the first 24 hours of admission (day 0) and through the days 1,2,4,6,8,10.

EBC collection at day 0 will be performed under both modalities of ventilation with the purpose to investigate the quantity and composition of the collected EBC from the same patient ventilated with different tidal volumes. For the next measurements, the EBC collection for each group will be performed under the preset conditions of ventilation.

Complications such as VAP, ARDS or sepsis during the period of observation will be recorded.

Statistical analysis:

Analysis will be performed using SPSS for Windows v. 16.0. Normality of distribution will be checked with Kolmogorov-Smirnov test. Normally distributed data will be presented as mean ± standard deviation (SD), whereas skewed data as median (interquartile range). Comparisons between two groups will be evaluated with unpaired t tests for normally distributed and Mann-Whitney tests for skewed data. Comparisons between more than two groups will be performed with analysis of variance (ANOVA) with appropriate post hoc tests

Inclusion Criteria:

• ICU patients mechanically ventilated because of stroke, subarachnoid and/or intracerebral hemorrhage
• Healthy respiratory system

Exclusion Criteria:

• Pulmonary diseases