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Supplementary materialThe effect of exhalation flow on endogenous particle emission and phospholipid composition Per Larsson, Bj?rn Bake, Anita Wallin, Oscar Hammar, Ann-Charlotte Almstrand, Mona L?rstad, Evert Ljungstr?m, Ekaterina Mirgorodskaya, Anna-Carin OlinInstrumental setup Normally, one mouthpiece with a two-way valve is used with the PExA instrument. Inhaled air is drawn through a HEPA-filter and exhalations are directed towards the PExA instrument where a second valve can be used to direct the exhaled air into the PExA instrument for sampling or back to ambient air. This configuration was not possible to use in the present study where maximal forced exhalations were studied. By removing the valve systems in front of the PExA instrument, it was possible to reduce the backpressure of the system and to achieve the maximal exhalation flow during exhalations into the PExA instrument. Furthermore, by exhaling into a straight, wide and open tube, the particle losses by impaction are kept to a minimum and the risk of non-endogenous particle formation is low. The modified PExA instrument used in the present study is described in figure S1. Participants were breathing particle free air through one mouthpiece and changed to a second mouthpiece connected to the PExA instrument only for the final exhalation of a breathing manoeuvre. In between exhalations into the PExA instrument, the mouthpiece opening was closed to prevent ambient particles from entering the instrument. The flow was visualized to the participant on a computer screen, both during inhalation and exhalation and this was used as visual feedback during breathing manoeuvres. Characterization of exhaled particlesParticle number concentrations in exhaled air were recorded by a Grimm 1.108 optical particle counter in eight different size channels at 1 Hz time resolution using the DUST MONITOR 3.30 software (Grimm Aerosol Technik GmbH & Co, Ainring, Germany). The size intervals of the particle counter were adjusted to 0.41–4.55 ?m in diameter since the optical properties of exhaled particles is different than the polystyrene latex spheres used for calibration by the manufacturer. The exhaled particle mass was calculated under the assumption that the exhaled particles were spherical in shape and had a density of 1.0 g·mL-1. The ability of a breathing manoeuvre to induce particle formation was measured as the emitted mass of particles per exhaled volume, i.e., mass concentration (pg·L-1).Sampling and chemical analysis of particlesThe mole amounts of DPPC and POPC in the extracted samples are presented in table S2 and are sorted by PEx mass. After sampling, the portion of the membrane where particles were deposited was cut out, placed in a 2.0 mL cryotube (Sarsteds, Nümbrecht, Germany), and stored dry at -20 °C for a maximum of 8 weeks before analysis. Sample extractionTo the samples 5 ?L internal standard (IS) containing 3 ?M of PC (17:0-20:4) and PC (17:0-14:1) (LM-1002 and LM-1004, Avanti lipids Alabaster, AL, USA) was added and allowed to dry. Samples were extracted using 160 ?L of a solvent consisting of methanol, chloroform, and 40 mM ammonium acetate (ratio 6:3:2, v/v/v) before centrifugation at 10?000*RCF for 2 minutes followed by shaking for 30 seconds. Sample analysisSamples were quantified using a triple quadrupole mass spectrometer (Sciex API300, Toronto, Canada) equipped with an electrospray ion source that was operated in positive mode at +5000 V and a turbo gas temperature of 375 °C. 20 ?L of sample was injected and run using a flow gradient method with an isocratic mobile phase of methanol, chloroform, and 40 mM ammonium acetate (ratio 6:3:2, v/v/v). The flow gradient was 50 ?L·min-1 for 0.1 min, 30 ?L·min-1 for 1.0 min and 200 ?L·min-1 for 0.2 min. Quantification of DPPC and POPC were performed with a selected reaction monitoring method using the transitions m/z 734.6>184.1 and 760.6>184.1 respectively. PC (17:0-14:1) was used as IS for DPPC quantification and PC (17:0-20:4) was used as IS for POPC quantification, using the transitions m/z 718.5>184.1 and 796.6>184.1 respectively. Standard samples were prepared in cryotubes containing PEx sampling membranes spiked with IS before adding a known amount of DPPC and POPC (850355C and 850457C, Avanti lipids Alabaster, AL, USA). Nine standards corresponding to an extracted amount between 0.16-80 picomole were prepared. From standards, a linear regression model using 1/x weighing where y=analyte area / IS area and x= analyte concentration/ IS concentration was constructed and used for calculating amount in unknown samples. The DPPC and POPC concentrations in particles were calculated as a weigh percent concentration (wt%) by dividing the extracted mass of DPPC and POPC from the sampling membrane with the total mass of sampled particles that was calculated from the data recorded by the optical particle counter. TABLE S1Characteristics of participantsNo.SexSmokingAge(yrs)Height (cm)Weight (kg)BMI(kg·m-2)FEV1(% pred)FVC(% pred)FEV1/FVC(% pred)1MNo361958522.4120121992MNo751737424.798107913FYes351687024.8113119954MNo281819829.98690965MNo531878524.392901016MNo651869326.91081011067FNo621666824.7102108948FNo281636424.18587989FNo311635018.8951008710MNo661827823.510310010311FNo461605521.511212490Participants individual values are presented. Predicted normal values (% pred) were calculated according to the ECCS/ERS equations. BMI: body mass index; FEV1 % pred: percent predicted of forced expiratory volume in one second; FVC % pred: percent predicted of forced vital capacity; PEF: peak expiratory flow. TABLE S2DPPC and POPC mole per sample and calculated weigh percent concentrations in particlesSubject IDManoeuvrePEx (ng)DPPC (picomole)DPPC (wt%)POPC ( picomole )POPC (wt%)1Reference 4.00.6211.50.112.29Reference 4.50.559.00.111.98Reference 5.60.354.70.111.54Reference 5.90.283.50.111.59Forced6.40.647.40.111.37Reference 6.90.576.00.111.27Forced8.20.504.50.111.02Reference 9.10.604.80.181.53Reference 9.31.3911.00.312.611Reference 10.01.279.30.302.34Forced13.50.110.60.110.69Cough15.60.110.50.110.67Cough15.90.180.80.110.58Airway reopening16.11.285.90.391.84Airway reopening18.62.8311.20.763.111Forced18.60.893.50.160.76Reference 20.91.786.20.481.810Cough22.61.685.50.381.39Airway reopening27.53.529.40.892.52Forced28.70.110.30.110.310Reference 30.24.1310.00.972.41Forced31.50.842.00.110.33Cough32.10.400.90.110.36Forced32.60.360.80.110.33Forced35.01.894.00.471.010Forced35.21.362.80.280.61Airway reopening40.76.7012.11.272.45Reference 42.04.708.21.342.48Cough46.00.110.20.110.25Cough50.82.964.30.771.211Cough53.61.141.60.270.42Airway reopening58.05.927.52.062.75Forced62.63.984.71.141.47Airway reopening68.85.896.31.541.78Forced70.10.260.30.110.16Airway reopening70.79.7610.12.672.911Airway reopening73.512.1412.13.073.23Airway reopening76.911.1210.62.902.96Cough82.00.950.80.230.21Cough100.20.310.20.110.12Cough125.40.270.20.110.15Airway reopening199.422.248.26.262.410Airway reopening318.741.609.610.702.64Cough434.81.320.20.380.1The mole amount of DPPC and POPC extracted are reported and sorted by increasing amount of sampled PEx mass. Concentrations below the LoQ of 0.16 picomole are shown with bold figures and the POPC samples that were excluded from the wt% dataset due to low PEx mass are shown with a darker shade of grey. Figure S1. Mouthpiece 1 (MP1): particle-free air was inhaled through a HEPA filter (a) whereas exhalations were directed to ambient air by a two-way valve. A flow meter (b) was installed next to the mouthpiece and connected to a computer to help the participant adhere to the target flows of the breathing manoeuvres. Mouthpiece 2 (MP2): the final expiration of a breathing manoeuvre was performed into the PEXA instrument. The exhaled aerosol was drawn through a two stage inertial impactor (c) with a constant volumetric flow of 230 mL·s-1 using a vacuum pump (d). The impactor 50 % cut-off was 7.0 ?m for the upper stage and 0.5 ?m for the lower stage. Exhaled particles are sampled on a membrane on the second impactor stage. The air drawn through the impactor was characterized in eight size intervals from 0.41to 4.55 ?m by an optical particle counter that operates at flow of 20 mL·s-1 (e). To handle exhalations exceeding the flow rate through the impactor, a reservoir (f) that can buffer the exhaled air was used. The reservoir was open at the bottom of the instrument (g) but was supplied with particle-free and humidified air (Respiratory Humidifier Fisher&Paykel MR 700) heated to 36 °C (h) at a flow rate of 280 mL·s-1; the supplied particle free air has a slightly higher volumetric flow than what is consumed by the vacuum pump and particle counter and the small over pressure generated prevents diffusion of ambient particles into the instrument in between exhalations. (i) A second flow meter was installed at the outlet of the breath reservoir to measure exhalation flow into the reservoir. All parts of the PEXA instrument, except the mouthpiece, are in a thermostated box at 36 °C (j).Figure S2. Mass fraction of particles in the interval 2.98–4.55 ?m in diameter out of the total interval 0.41–4.55 ?m plotted against peak expiratory flow for a) the forced manoeuvre and b) the cough manoeuvre. The observations are numbered according to identification numbers in extended table 1. PEF: peak expiratory flow; %: percent of total particle mass between 0.41–4.55 ?m in diameter; L?min-1: litres exhaled breath per minute; rs: Spearman correlation.Figure S3. DPPC to POPC mole ratios for each individual. The five replicate measurements on subject 1 are presented on the far right in the figure. ................
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