Phospholipid composition and kinetics in different endobronchial fractions from healthy volunteers

Continued synthesis and secretion of pulmonary surfactant is critically important for maintenance of optimal lung function throughout life. While primary surfactant deficiency in the lungs of preterm infants is widely acknowledged as a major cause of neonatal respiratory distress syndrome, secondary surfactant deficiency contributes to the pathology of many respiratory disorders of the mature lung including acute lung injury (ALI)/ acute respiratory distress syndrome (ARDS), asthma and cystic fibrosis [1]. Described mechanisms for surfactant dysfunction in respiratory diseases include inhibition by plasma proteins such as fibrinogen in oedema fluid, impaired synthesis and secretion by type II alveolar epithelial (ATII) cells and hydrolysis or oxidation of secreted surfactant [2]. Differentiating between these potential mechanisms is problematical in the clinical setting. Acquiring samples of alveolar and/or bronchial secretions for analysis of surfactant function or composition typically involves invasive bronchoscopic and bronchoalveolar lavage (BAL) procedures [3]. Additionally, analysis of concentrations and compositions of surfactant components such as surfactant proteins or phospholipids provides no information about processes of surfactant synthesis, secretion or turnover [4, 5]. Availability of such information has considerable potential clinical implications, both for better understanding mechanisms of lung disease and for optimizing treatments for individual patients. Consequently, in this study we have assessed the molecular specificity of surfactant phospholipids extracted from small volume bronchoalveolar lavage fluid (BALF), tracheal wash (TW) and induced sputum (IS), representing secretions from various endobronchial compartments. In addition, we employed an in vivo stable isotope labelling strategy to monitor the incorporation of methyl-D₉-choline chloride into the phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) fractions of the three different sample types. PC, especially the disaturated dipalmitoyl species (PC16:0/16:0), is the major surface active component of surfactant phospholipid. This stable isotope methodology enables the assessment of both the rate of synthesis and secretion of individual molecular species of surfactant PC and the specificity of fatty acyl remodelling mechanisms involved in their synthesis. This detailed analysis not only provides important information about mechanisms of surfactant PC synthesis and secretion, but comparison of fractional incorporation rates between sample types can also demonstrate the time required for newly secreted alveolar surfactant to transit to the upper airways.

Ten healthy volunteers were recruited. All participants had sputum induction. One subject, however, was unable to tolerate bronchoscopy leaving BALF/TW analysis for the remaining nine participants. The clinical data and summary of the participant characteristics were listed in Table 1.

This study demonstrates for the first time the molecular compositions of various bronchoalveolar compartments in human model and the surfactant molecular PC kinetics from all these compartments. The results showed that surfactant extracted by TW closely resembled that of BALF. As ATII cells are the only source of pulmonary surfactant phospholipids, the significant positive correlation of methyl-D₉-choline incorporation between BALF and TW suggests the mucocillary transit time of surfactant up along the airways had little significant impact on surfactant PC kinetics. Our analysis suggests that, despite the presence of already-secreted surfactant lining the small and large airway epithelium, a surfactant pool accessible from the upper airways by tracheal wash is in isotopic equilibrium with the alveolar surfactant pool.

Induced sputum had variable phospholipid composition and kinetics compared to BALF, possibly due to several reasons. Firstly, IS phospholipids may represent pools of surfactant with different temporal origins, with some material newly secreted from the alveolus combined with previously secreted surfactant embedded within the mucus layer. Secondly, sputum induction is a complex process and the exact origin of the induced sputum is not always known. Furthermore, the contamination from saliva and other non- surfactant phospholipids may have interfered with surfactant assessment. Although IS had qualitatively similar composition to that of alveolar surfactant [10], it may not be an ideal model to study alveolar surfactant metabolism. Thirdly, the relatively high content of sphingomyelin in induced sputum suggests proportion of IS phospholipid may be of cellular not surfactant origin. Such a cellular origin of IS phospholipid may become more significant in disease states characterized by increased airway inflammatory cell infiltration.

Surfactant PC is synthesized de-novo by the CDP-choline pathway. However, about 50% of PC16:0/16:0 is produced by acyl-remodelling mechanisms catalysed by the sequential actions of phospholipase-A₂ and lysophosphatidylcholine acyltransferase activities [11]. It is widely assumed that surfactant PC synthesis and acyl-remodelling processes precede subsequent secretion [11]. However, our finding challenges this established concept. If the surfactant PC is secreted after acyl-remodelling and maturation process, the secreted surfactant PC16:0/16:0 compositions should reflect the endogenous composition at all time points. Our study shows that the proportion of methyl-D₉-labelled PC16:0/16:0 is equilibrated with endogenous composition only at 48 hours. This implies that a certain proportion of PC is secreted even before the acyl-remodelling mechanisms are complete.

This is the first to study to demonstrate the feasibility of assessing surfactant lyso-PC metabolism in-vivo in human subjects. This study shows that LPC16:0 is the principle lyso-PC in pulmonary surfactant, but that total LPC methyl-D₉ incorporation was twice as that of total PC. The methyl-D₉-incorporation of LPC16:0 is much higher than that of PC16:0/16:0 at both 24 and 48 hours. This finding challenges the paradigm of that the LPC is a formed by hydrolysis of secreted surfactant PC. The much higher fractional incorporation of LPC compared to PC precludes LPC coming from hydrolysis of newly synthesized PC and emphasizes the complexity of the underlying mechanisms of surfactant synthesis and secretion. These observations strongly support the possibility, at least in healthy individuals, that LPC is secreted together with the other surfactant phospholipid rather than simply being a consequence of hydrolysed PC. While phospholipase-mediated hydrolysis of secreted surfactant phospholipid remains a possibility in inflammatory lung disease, a further implication of our results, supported by the strong correlation of stable isotope label incorporations into LPC16:0 and PC16:0/16:0 (Figure 4) is that secreted LPC may be ultimately a consequence of acyl remodelling mechanisms within ATII cells.

Investigators have also used other stable isotopes such as ¹³C-glucose, ¹³C-fatty acids or deuteriated water to assess fractional synthetic rates (FSR) of saturated PC (SatPC) to characterise surfactant kinetics in neonates [12‐14] and adults [15]. SatPC is the major PC surfactant component and a clinical marker of lung injury and surfactant derangement [16]. Measuring SatPC from deuteriated water enables maintenance of a steady state condition more easily, and therefore results can be compared between groups of patients with different clinical conditions. These novel studies provided significant insights into surfactant metabolism in humans but have some limitations. First, these methodologies initially used osmium tetroxide oxidation of unsaturated phospholipid to generate a SatPC fraction. Consequently, minimal information can be obtained about the molecular specificity of surfactant PC metabolism [17]. Second, the laborious sample preparation required for these studies has precluded the early diagnostic application of these methodologies to individuals with lung diseases. In contrast, the use of ESI-MS to monitor stable isotope incorporations requires minimal sample preparation and has the potential to generate diagnostic surfactant kinetic results in a time scale consistent with making clinical treatment decisions for individual patients with acute respiratory compromise.

In our study, tracheal secretions were accessed by the use of a bronchoscope. It is entirely plausible that a small amount of the BALF from distal alveolus may have contaminated the tracheal washings as both of these procedures were conducted in a single setting. If the compositional similarities were due to this contamination, one might have seen significant dilution effect in the magnitude of 1:10 reduction in the fractional phospholipid concentrations from tracheal wash. However, this was not the case and this absence of dilution effect indicate any contamination of tracheal wash from alveolar material is minimal.

Another limitation of this study was the measurements of absolute amounts of phospholipid subclasses and PC species may have been influenced by the variability in the sample recovery. Nevertheless it was reassuring to see overall compositional similarities from all endobronchial sampling. Furthermore, kinetic data regarding methyl-D₉ incorporation patterns are unlikely to be influenced by this variability in recovery as the incorporation data was corrected for the endogenous material.

Acute lung injury and ARDS are characterized by significant quantitative and qualitative alterations in surfactant phospholipids composition [5]. Despite these findings, therapeutic attempts with exogenous surfactant remain unhelpful in this population [18]. The complexity of ARDS pathogenesis suggests several possible mechanisms for surfactant dysfunction, such as impaired synthesis and secretion, increased hydrolysis, proteolysis and oxidation or functional inhibition by infiltrating plasma proteins. However, in-vivo human models investigating such complex underlying mechanisms are lacking. Deficient understanding of the potential multiple mechanisms of aberrant surfactant metabolism in this disease cohort may in part explain the lack of anticipated clinical benefits from exogenous surfactant replacement strategies. Application of stable isotope studies in this population may possibly identify underlying phenotypes to characterise patients according to pathological mechanisms of surfactant dysfunction. Tracheal washings substituted for quantitative bronchoalveolar lavage may be an alterative for surfactant isolation in patients otherwise unable to tolerate invasive procedures without clinical compromise.