Metabolism of a synthetic compared with a natural therapeutic pulmonary surfactant in adult mice

Abstract

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.

Keywords: acyl remodeling; mass spectrometry; molecular species; phosphatidylcholine synthesis; stable isotopes.

Fig. 1.

PC molecular species analysis of the mouse lung and exogenous surfactants. Compositions of mouse surfactant (A), poractant alfa (B), and CHD5633 (C) detailed by precursor scanning of the phosphocholine ion fragment at m/z 184. Precursor scanning of the [5¹³C]phosphocholine head group confirmed that [U¹³C]DPPC was >99% isotopically labeled (D).

Fig. 2.

Time dependence of exogenous surfactant DPPC enrichment in mouse lung BALF (A) and in the lavaged lung (B). Results were calculated by normalizing [U¹³C]DPPC enrichments for the ¹³C enrichment of DPPC in the administered surfactant. Results are presented as means ± SEMs. *P < 0.05, ^†P < 0.01, and ^‡P <0.001, unpaired t-test.

Fig. 3.

Effect of exogenous surfactant on the synthesis of endogenous DPPC by the lungs of mice administered CHF5633 (A) or poractant alfa (B). The enrichment of [D₉]choline in endogenous DPPC was determined after correcting for the concentration of exogenous DPPC. Mice were divided at each time point into two equal groups based on the variation of exogenous surfactant enrichment in BALF. Any inhibition of PC synthesis by exogenous surfactant would have been seen by lower [D₉]choline DPPC enrichment in the group of mice that received a higher dose of surfactant.

Fig. 4.

Time dependence of [U¹³C]DPPC and [D₉]DPPC enrichment in the lungs (A, B) and BALF (C, D) for mice administered CHF5633 (A, C) or poractant alfa (B, D). Results for [U¹³C]DPPC were normalized for stable isotope enrichments of administered surfactants to enable a comparison between CHF5633 and poractant alfa. Results are presented as means ± SEMs.

Fig. 5.

Precursor ion scanning of labeled BALF PC. A: Precursor scan of m/z 193 detailing the incorporation of methyl-D₉-choline into PC species 9 amu greater than unlabeled PC. B: Precursor ion scan of m/z 189 showing the incorporation into PC species of [5¹³C]choline derived from [U¹³C]DPPC. The incorporation of [5¹³C]choline by the CDP-choline pathway is given by odd-numbered species 4 amu lower than in A (e.g., m/z 739.5 vs. 743.6). The addition of an unlabeled acyl chain to [24¹³C]lysoPC16:0 is indicated by even-numbered species (e.g., m/z 806.6 and 830.6). These spectra are from a mouse 48 h after being administered CHF5633 and methyl-D₉-choline.

Fig. 6.

Incorporation of labeled choline into PC molecular species of the mouse lung (A, B, C, and F) and BALF (D, E, and G). Incorporation of [5¹³C]choline derived from the hydrolysis of [U¹³C]DPPC is shown for the enrichments of total [5¹³C]PC species in the lungs (A), individual [5¹³C]PC species in the lungs from CHF5633- (B) and poractant alfa-treated mice (C), and individual [5¹³C]PC species in BALF from CHD5633- (D) and poractant alfa-treated mice (E). Results were normalized for the enrichment of [U¹³C]DPPC in administered surfactant to enable a direct comparison between CHF5633- and poractant alfa-treated mice. The incorporation of methyl-D₉-choline is shown in F and G for the lungs and BALF for CHF5633-treated mice. Results are presented as means ± SEMs.

Fig. 7.

Selectivity of acyl remodeling of [24¹³C]lysoPC16:0 in mouse lungs for CHF5633 (A) and poractant alfa (B). [24¹³C]lysoPC16:0 was generated by the action of phospholipase A₂ on [U¹³C]DPPC, followed by reacylation with an unlabeled [¹²C]acyl residue. Results are shown for percentage label enrichments as means ± SEMs for [24¹³C]PC16:0/20:4 and [24¹³C]PC16:0/22:6 and only as errors for [24¹³C]PC16:0/16:0 and [24¹³C]PC16:0/18:1. All other PC species were too small to display.

Fig. 8.

Recycling of [U¹³C]DPPC. Neutral loss scans illustrating the metabolic routes whereby [U¹³C]DPPC is metabolized and labeled fragments are recycled into BALF DPPC. The scans were derived from a mouse that received [U¹³C]DPPC-labeled CHF5633 for 24 h. A: Unlabeled DPPC. B: M+3 isotopomer of DPPC and potentially [¹³C]glycerol-labeled DPPC. C: Incorporation of one [¹³C]palmitoyl group into DPPC. D: Reacylation of [24¹³C]lysoPC16:0 with an unlabeled [¹²C]palmitoyl group. E: Combination of two [¹³C]palmitoyl groups with unlabeled [¹²C]glycerophosphocholine (m/z 766.7) and M-3 isotopomer of [U¹³C]DPPC (m/z 771.8). F: [U¹³C]DPPC.

Fig. 9.

Recycling of [¹³C]palmitate into BALF DPPC by mouse lungs in vivo. Neutral loss scans of m/z 567 and 583 detected the incorporation of [¹³C]palmitate derived from [U¹³C]DPPC back into, respectively, ([¹³C]palmitoyl)-DPPC ([16¹³C]PC16:0/16:0; m/z750) and (di-¹³C-palmitoyl)-DPPC ([32¹³C]PC16:0/16:0; m/z 766) in BALF from mice administered either CHF 5633 (A) or poractant alfa (B). The corresponding neutral loss scan of m/z 570 details the acylation of [24¹³C]lysoPC16:0 with unlabeled palmitate to form ([¹²C]palmitoyl)-[24¹³C]DPPC ([24¹³C]PC16:0/16:0; m/z 758) and DPPC containing both labeled palmitate and glycerol ([¹³C]palmitoyl and [¹³C]glycerol-DPPC; m/z 753). Results are presented as means ± SEMs. C: Precursor scans of palmitoyl (m/z 255) and [16¹³C]palmitoyl (m/z 271) into PC species in negative ionization. PC species were detected as M-16⁻ ions. This spectrum was at 72 h after surfactant administration, which is why no ion peak for [U¹³C]DPPC-16⁻ was observable at m/z 758.6.