Markers in Exhaled Breath Condensate in Obstructive Sleep Apnoea (OSA) Patients

Measurement of exhaled Nitric Oxide and collection of exhaled breath condensate and peripheral blood. This will be done 2 to 4 times(on admission,the morning after sleep study,immediately after CPAP titration, after CPAP use for 3 months). Each time eNO will be measured first, followed by EBC collection. For the tests in the morning, patients will be asked to postpone his/her breakfast until after the test which will take less than half an hour. OSA is defined as patients having a respiratory disturbance index of ≥5/hr. Measurement of Exhaled Nitric Oxide: eNO will be measured using a Sievers 280i chemiluminescence analyser (Sievers Instruments, Boulder, CO, USA) sensitive to NO from 1 ppb to 200 ppm and with a resolution of 1 ppb and accuracy of 1 ppb designed for online recording. The eNO measurement will be performed according to ATS standards. Briefly, subjects should remain seated without nose clip during the procedure, with 2-minute rest between eNO measurements. The eNO will be measured online at an expiratory flow rate of 50 ml/sec, a dead space of 10 ml and 2-second plateau duration. Repeated exhalations are performed until three NO plateau values agree at the 10% level or two agree at the 5% level. The mean NO value will then be recorded. Collection of Exhaled Breath Condensate: EBC will be collected using the ECoscreen (Jaeger, Germany) according to manufacturer instructions. After rinsing their mouth, the recruited subjects will breathe tidally through a mouthpiece that is connected through a unique one-way valve into a cooled collection tube where vapours, aerosols and moisture in the breath condense along the walls of the tube. The one-way valve is then used as a plunger that collects droplets stuck to the inside wall and holds the sample near the top of the tube. The device inherently prevents salivary contamination as shown by the absence of salivary amylase in EBC. On average, this device will be able to collect 300 microL per minute of EBC for an adult. Each subject will be asked to breathe through the collection kit for 5 minutes, which should be sufficiently long to yield 1 ml of condensate for inflammatory marker analysis. EBC will be stored immediately at -70oC until analysis for inflammatory markers. Measurement of 8 isoprostane, GRO alpha, MCP1, eotaxin and MDC levels in exhaled breath condensate: EBC stored at -70oC will be analysed for 8-isoprostane, GROalpha, MCP1, eotaxin and MDC levels concentrations in batches. We will use 96-well polystyrene microplates coated with murine monoclonal antibodies against human 8 isoprostane, GRO alpha, MCP1, eotaxin and MDC levels. The levels of these markers in EBC will be measured in duplicates by sandwich enzyme immunoassay (R \& D Systems, Minneapolis, MN, USA) according to instructions provided by the manufacturer.