Marine mammals are repeatedly exposed to elevated extra-thoracic pressure and alveolar collapse during diving and readily experience alveolar expansion upon inhalation – a unique capability as compared to terrestrial mammals. in odontocetes whose physiology and diving behavior differ from pinnipeds. The aim of this study was to investigate the phosphatidylcholine (PC) composition of lung surfactants from various marine mammals and compare these to a terrestrial mammal. We found an increase in anti-adhesive PC species in harp seal (ID: IFAW 12 208Pg) or stranded – California sea lion (ID: D08335Dd) and harbor porpoise (Phocoena phocoena; IDs: D08091Pp Pp1201_Theo C-386_Pp) – and were obtained from the National Oceanic and Atmospheric Administration National Marine Fisheries Service Northeast Fisheries Observer Program the Marine Mammal Rescue and Research Division of the International Fund for Animal Welfare the Marine Mammal Center (Sausalito CA) or the Vancouver Aquarium (Vancouver BC). The time of death was known for the stranded specimens and the approximate time was recorded for bycaught specimens. All specimens were in good post mortem condition (Smithsonian Institute code 2; Geraci and Lounsbury 2005 and none were decomposed when the lavage sample was obtained. The lavage fluid was spun at 200 g for 15 minutes and the supernatant was collected. The supernatant was spun at 39 0 g for 60 minutes and the pellet was resuspended in isotonic saline solution and frozen at ?80°C until further analysis. Where indicated the lavage fluid was frozen prior to surfactant isolation (designated as frozen) or left at 4°C or room temperature for up to two days (timecourse analyses). 2.2 Phospholipid extraction Aliquots (1 mL) of resuspended surfactant samples were mixed with 2:1 chloroform:methanol to achieve a final solvent ratio of 8:4:3 chloroform:methanol:isotonic saline (Folch 1956). Samples were vortexed for one minute and centrifuged at 930 g for 5 minutes. The bottom chloroform layer was collected and dried under a stream of N2. Samples were stored dessicated at ?20°C. 2.3 Mass spectrometry analysis Immediately prior to analysis samples were reconstituted in 1 mL of methanol. An aliquot of each sample (100 μL) was transferred to an autosampler vial and methanol diluted (10-fold) or undiluted samples were placed in the autosampler of an Agilent 1200 HPLC (Agilent Technologies Rabbit Polyclonal to PDZD2. Santa Clara CA) at 20°C and 5 μL was injected. Samples were filtered through a column shield (MAC-MOD Analytical Chadds Ford PA) onto a Kinetex C18(2) 150 × 3 mm 2.6 μm 100 ? column (Phenomenex Torrance CA) at 30°C. Phospholipids were separated isocratically at a 0.4 mL/min flow rate using a mobile phase mixture of 95% methanol 5 water and 10 mM ammonium acetate for 40 minutes. Samples were analyzed via electrospray ionization in positive ion mode using an Agilent 6410B triple quadrupole mass spectrometer with MassHunter Data Acquisition software (version B.02.01 Agilent Technologies). For qualitative analysis samples were analyzed in full scan mode with a mass-to-charge (m/z) range of 600-850 and a fragmentation voltage of 200. To identify specific PCs product ion scans were performed in negative ion mode on the acetate adducts. Additionally precursor ion scans for m/z 184 Flecainide acetate were acquired in positive ion mode to confirm the presence of phosphatidylcholine. For quantitative analysis samples were analyzed via multiple reaction monitoring. Optimal transition ions were determined using standards and MassHunter Optimizer software (ver B.02.01 Agilent Technologies). Fragmentor voltages and transition ions for the quantified PC phospholipids are listed in Supplementary Table 1. Data were analyzed using Qualitative Analysis software (ver. B.03.01 Agilent Technologies). Sodiated adducts were not taken into account for quantitation. 2.4 Identification and Quantitation Retention time m/z and MS/MS of standard PCs were used to confirm the identity of sample PCs. PCs in each sample were quantified by comparing peak area in the samples to PC standard curves obtained by analysis of a mixture of PC phospholipids at seven different concentrations ranging Flecainide acetate from 0.2 – 20 ng/μL in duplicate or triplicate. Each sample PC identified was compared to the same standard PC with the exception of PC 16:1/18:1 which was quantified using the PC 16:0/18:2 standard and PC 16:0/16:1 which Flecainide acetate was quantified using the PC 16:0/16:0 standard. These alternative standards were used as replacements because PC 16:1/18:1 and 16:0/18:2 were not readily obtainable. For a small number of samples (6%) the calculated injected sample Flecainide acetate concentrations were outside of the 0.2 – 20 ng/μL range.