Secreted pulmonary surfactant phosphatidylcholine (PC) has a complex intra-alveolar metabolism that involves uptake and recycling by alveolar type II epithelial cells, catabolism by alveolar macrophages, and loss up the bronchial tree. We compared the in vivo metabolism of animal-derived poractant alfa (Curosurf) and a synthetic surfactant (CHF5633) in adult male C57BL/6 mice. The mice were dosed intranasally with either surfactant (80 mg/kg body weight) containing universally 13C-labeled dipalmitoyl PC (DPPC) as a tracer. The loss of [U13C]DPPC from bronchoalveolar lavage and lung parenchyma, together with the incorporation of 13C-hydrolysis fragments into new PC molecular species, was monitored by electrospray ionization tandem mass spectrometry. The catabolism of CHF5633 was considerably delayed compared with poractant alfa, the hydrolysis products of which were cleared more rapidly. There was no selective resynthesis of DPPC and, strikingly, acyl remodeling resulted in preferential synthesis of polyunsaturated PC species. In conclusion, both surfactants were metabolized by similar pathways, but the slower catabolism of CHF5633 resulted in longer residence time in the airways and enhanced recycling of its hydrolysis products into new PC species.

The Bill & Melinda Gates Foundation funded the study, grant number INV-016631. The NIHR Southampton Biomedical Research Centre provided mass spectrometry facilities and salary support for GK and MP. The NIHR Southampton Biomedical Research Centre provided mass spectrometry facilities and salary support for GK and MP. MPWG receives part of his funding through the NIHR Senior Investigator Scheme. RD and MPWG received part of their funding through the NIHR Southampton Biomedical Research Centre. H.W.C. and D.B. received part of their funding through the

Mass spectrometry imaging (MSI) visualizes molecular distributions throughout tissues but is blind to dynamic metabolic processes. Here, MSI with high mass resolution together with multiple stable isotope labeling provided spatial analyses of phosphatidylcholine (PC) metabolism in mouse lungs. Dysregulated surfactant metabolism is central to many respiratory diseases. Metabolism and turnover of therapeutic pulmonary surfactants were imaged from distributions of intact and metabolic products of an added tracer, universally 13 C-labeled dipalmitoyl PC (U 13 C-DPPC). The parenchymal distributions of newly synthesized PC species were also imaged from incorporations of methyl -D 9 -choline. This dual labeling strategy demonstrated both lack of inhibition of endogenous PC synthesis by exogenous surfactant and location of acyl chain remodeling processes acting on the U 13 C-DPPC-labeled surfactant, leading to formation of polyunsaturated PC lipids. This ability to visualize discrete metabolic events will greatly enhance our understanding of lipid metabolism in diverse tissues and has potential application to both clinical and experimental studies.