Altered molecular specificity of surfactant phosphatidycholine synthesis in patients with acute respiratory distress syndrome

Ahilanandan Dushianthan, Victoria Goss, Rebecca Cusack, Michael P W Grocott, Anthony D Postle, Ahilanandan Dushianthan, Victoria Goss, Rebecca Cusack, Michael P W Grocott, Anthony D Postle

Abstract

Background: Acute respiratory distress syndrome (ARDS) is a life-threatening critical illness, characterised by qualitative and quantitative surfactant compositional changes associated with premature airway collapse, gas-exchange abnormalities and acute hypoxic respiratory failure. The underlying mechanisms for this dysregulation in surfactant metabolisms are not fully explored. Lack of therapeutic benefits from clinical trials, highlight the importance of detailed in-vivo analysis and characterisation of ARDS patients according to patterns of surfactant synthesis and metabolism.

Methods: Ten patients with moderate to severe ARDS were recruited. Most (90%) suffered from pneumonia. They had an infusion of methyl-D9-choline chloride and small volume bronchoalveolar lavage fluid (BALF) was obtained at 0,6,12,24,48,72 and 96 hours. Controls were healthy volunteers, who had BALF at 24 and 48 hours after methyl-D9-choline infusion. Compositional analysis and enrichment patterns of stable isotope labelling of surfactant phosphatidylcholine (PC) was determined by electrospray ionisation mass spectrometry.

Results: BALF of patients with ARDS consisted of diminished total PC and fractional PC16:0/16:0 concentrations compared to healthy controls. Compositional analysis revealed, reductions in fractional compositions of saturated PC species with elevated levels of longer acyl chain unsaturated PC species. Molecular specificity of newly synthesised PC fraction showed time course variation, with lower PC16:0/16:0 composition at earlier time points, but achieved near equilibrium with endogenous composition at 48 hours after methyl-D9-choline infusion. The enrichment of methyl-D9-choline into surfactant total PC is nearly doubled in patients, with considerable variation between individuals.

Conclusions: This study demonstrate significant alterations in composition and kinetics of surfactant PC extracted from ARDS patients. This novel approach may facilitate biochemical phenotyping of ARDS patients according to surfactant synthesis and metabolism, enabling individualised treatment approaches for the management of ARDS patients in the future.

Figures

Figure 1
Figure 1
Bronchoalveolar lavage phosphatidylcholine mass spectra from a healthy control for precursor scans of P184 (A) (endogenous phosphatidylcholine composition) and precursor scans of P193 (B) (newly synthesised deuterated phosphatidylcholine composition at 24 hours aftermethyl-D9choline infusion). The relative signal intensity of spectra B is nearly one hundredth of spectra A. The m/z 678 represents an internal standard of PC14:0/14:0. The methyl-D9-labelled phosphatidylcholine composition consisted of phosphatidylcholine species with 9 mass units higher than that of endogenous composition. PC, phosphatidylcholine; m/z, mass to charge ratio.
Figure 2
Figure 2
Bronchoalveolar lavage total phosphatidylcholine concentrations over time for patients and controls. Measured by sum of all phosphatidylcholine species present >1% abundance. [Controls (n) =9 for both time points, patients (n) =10, n = 9, n = 10, n = 9, n = 9, n = 8, n = 7 for time points 0,6,12,24,48,72 and 96 respectively]. Data expressed as mean ± SEM. BALF, bronchoalveolar lavage fluid; PC, phosphatidylcholine.
Figure 3
Figure 3
Comparison of bronchoalveolar lavage surfactant phosphatidylcholine composition between patients at enrolment (T = 0 Hrs) and controls at earliest time point after enrolment (T = 24 Hrs). Controls (n) =9 and patients (n) =10. Presented as mean ± SEM, †, P < 0.0001; ‡, P < 0.05. PC, phosphatidylcholine.
Figure 4
Figure 4
Individual variations of surfactant specific phosphatidylcholine species (PC16:0/14:0), PC16:0/16:1 and PC16:0/16:0) between controls at earliest time point after enrolment (T=24 Hrs) (A), and patients at enrolment (T=0) (B). Controls (n) =09 and patients (n) =10. PC, phosphatidylcholine; C, individual controls; P, individual patients.
Figure 5
Figure 5
Comparison of total bronchoalveolar lavage surfactant phosphatidylcholine (A) and fractionalmethyl-D9PC16:0/16:0 (B), PC16:0/14:0 (C), PC16:0/16:1 (D), PC16:0/18:2 (E) and PC16:0/18:1 (F) enrichment in patients and controls. Data presented as mean ± SEM, †, P < 0.05. PC, phosphatidylcholine.
Figure 6
Figure 6
Bronchoalveolar lavage surfactant total phosphatidylcholine and fractional PC16:0/16:0 enrichment patterns for individual patients and controls. A, Individual variation in the total PC methyl-D9 enrichment among patients; B, Individual variation in the PC16:0/16:0 methyl-D9 enrichment among patients; C, Individual variation in the total PC methyl-D9 enrichment among controls; D, Individual variation in the PC16:0/16:0 methyl-D9 enrichment among controls.
Figure 7
Figure 7
Fractional synthetic rate (FSR) of phosphatidylcholine enrichment in BALF samples from patients with ARDS. Results were calculated as the initial gradient of enrichment divided by the plateau enrichment expressed as a percentage. While there was a three-fold range in values, there was no significant difference for those who survived (mean = 1.64 h−1) and those patients who died (mean = 1.40 h−1, P = 0.36).
Figure 8
Figure 8
Molecular specificity of newly synthesised major bronchoalveolar lavage surfactant phosphatidylcholine fraction in ARDS patients over time (A), endogenous andmethyl-D9labelled fractional composition of PC16:0/16:0 for both controls and patients (B), endogenous andmethyl-D9labelled fractional composition of PC16:0/16:0 for individual controls (C) and patients (D) at 48 hours aftermethyl-D9-choline infusion. At the earliest time point (T = 06 Hrs) after methyl-D9 choline infusion, unsaturated PC species predominate with subsequent gradual increase in PC16:0/16:0 over time. PC16:0/16:0 synthesis, expressed as a percentage of newly-synthesised total PC, was compared with that of endogenous PC16:0/16:0 at two time points for controls and six time points for patients. The proportion of methyl-D9-labelled PC16:0/16:0 was significantly lower than that of unlabelled PC16:0/16:0, when both are expressed relative to total labelled and unlabelled PC pools, and at 24 hours this deficit is greater in ARDS compared with control subjects. For both patients and controls, the newly synthesised fraction of PC16:0/16:0 was in equilibrium with endogenous composition at 48 hours after methyl-D9 choline infusion. Presented as mean ± SEM; † P < 0.05. There is considerable variation in the relative proportion of fractional labelling of PC16:0/16:0 in patients compared to the controls in addition to lower fractional synthesis of PC16:0/16:0 in 3 patients (P02, P04 and P08) compared with endogenous fraction. Controls (n) =9 and patients (n) =9. PC, phosphatidylcholine. C, individual controls; P, individual patients.

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