Abnormal liver phosphatidylcholine synthesis revealed in patients with acute respiratory distress syndrome

Abstract

Acute respiratory distress syndrome (ARDS) is associated with a severe pro-inflammatory response; although decreased plasma cholesterol concentration has been linked to systemic inflammation, any association of phospholipid metabolic pathways with ARDS has not been characterized. Plasma phosphatidylcholine (PC), the major phospholipid of circulating lipoproteins, is synthesized in human liver by two biologically diverse pathways: the cytidine diphosphocholine (CDP):choline and phosphatidylethanolamine N-methyltransferase (PEMT) pathways. Here, we used ESI-MS/MS both to characterize plasma PC compositions and to quantify metabolic fluxes of both pathways using stable isotopes in patients with severe ARDS and in healthy controls. Direct incorporation of methyl-D₉-choline estimated CDP:choline pathway flux, while PEMT flux was determined from incorporations of one and two methyl-D₃ groups derived from methyl-D₉-choline. The results of MS/MS analysis showed significant alterations in plasma PC composition in patients with ARDS versus healthy controls. In particular, the increased overall methyl-D₉-PC enrichment and, most importantly, the much lower methyl-D₃-PC and methyl-D₆-PC enrichments suggest increased flux through the CDP:choline pathway and reduced flux through the PEMT pathway in ARDS. To our knowledge, this study is the first to demonstrate significant plasma PC molecular compositional changes combined with associated alterations in the dynamics of PC synthetic pathways in patients with ARDS.

Keywords: S-adenosyl methionine; methyl-D9-choline; phosphatidylethanolamine N-methyltransferase; stable isotope.

Fig. 1.

Plasma concentrations (mmol/l) of choline (A), PC (B), total cholesterol (C), HDL-cholesterol (D), LDL-cholesterol (E), and triacylglycerol (F) between ARDS patients and healthy controls. Plasma choline and PC were measured for the duration of the investigative period (4 days). Cholesterol and triacylglycerol concentrations are at time t = 0 on day 1. Error bars indicate SEM. Statistical analysis unpaired Student’s t-test. ¥, P < 0.05.

Fig. 2.

Concentration of selected plasma PC molecular species at enrollment (t = 0). A: Mean distribution of PC species. Error bars indicate SEM. Statistical analysis unpaired Student’s t-test. ¥, P < 0.05. B–E: Scatter plots to illustrate the variation and differences between patient and control groups for selected major (>2% presence) PC species containing 18:2 (B), PC18:1 (C), PC20:4 (D), and PC22:6 (E).

Fig. 3.

Plasma methyl-D₉-choline (A) and methyl-D₉-betaine (B) enrichments over the total investigative period (4 days). The earliest measured methyl-D₉-choline enrichment, expressed as a percentage of total plasma choline, was 10% and 8% for patients and controls, respectively, with a corresponding value of 9% for methyl-D₉-betaine from both groups. Turnovers of methyl-D₉-choline (P < 0.05) enrichment were faster for patient (solid line) compared with control (dashed line) groups (mean ± SEM). A similar trend was observed for methyl-D₉-betaine, although this was not statistically significant. Half-lives were calculated from exponential fits to the data.

Fig. 4.

Schematic illustration of PC synthesis and incorporation of labeled methyl-D₉-choline via the CDP:choline and PEMT pathways. Deuterated methyl groups are highlighted as red. The fate of each deuterated methyl group can be traced in individual PC species synthesized by both pathways. CK, choline kinase; CT, phosphocholine cytidylyltransferase; CPT, choline phosphotransferase.

Fig. 5.

Methyl-D₉-PC (A), methyl-D₃-PC (B), methyl-D₆-PC (C), and methyl-D₃-SAMe (D) enrichment between patients and controls. The PC enrichment was calculated as percentage of total plasma PC pool. Methyl-D₃-SAMe was estimated by P190/Σ(P187 + P190). The solid line represents patients and the dashed line corresponds to controls. Data are expressed as mean ± SEM. ¥, P < 0.05. Half-lives for SAMe were calculated from exponential fits to the data and were significantly different (P < 0.05) for patient (3.4 h) and control (18.3 h) groups.

Fig. 6.

Fractional methyl-D₉-PC (A, B), methyl-D₃-PC (C, D), and methyl-D₆-PC (E, F) enrichment for individual PC species. Results are presented as the labeled fraction in the total plasma PC pool for that individual PC species, compared between patients and control groups over the investigative period for 4 days. Each line color represents individual PC species; blue, PC16:0_18:2; red, PC16:0_18:1; green, PC16:0_20:4; purple, PC16:0_22:6; yellow, PC18:0/20:4; and black, PC18:0/22:6. Data are expressed as mean ± SEM.

Fig. 7.

Fractional synthesis of PC via the PEMT pathway. This was estimated by using total methyl-D₃-SAMe corrected for the corresponding methyl-D₃-PC enrichment and presented as the percentage of total PC synthesis. The solid line represents patients and the dashed line corresponds to controls, both calculated as sigmoidal curve fits (P < 0.05).

Fig. 8.

Molecular specificity of PC fractional synthesis via the PEMT pathway. This was estimated by using methyl-D₃-SAMe corrected for the corresponding methyl-D₃-PC enrichment for the particular PC species of interest. Among the selected PC species, PC16:0_22:6 followed by PC16:0_22:6 were the major PC species synthesized by this pathway. Each colored line represents individual molecular PC species: blue, PC16:0_18:2; red, PC16:0_18:1; green, PC16:0_20:4; purple, PC16:0_22:6; and yellow, PC18:0_22:6. Data are expressed as mean ± SEM.

Fig. 9.

Plasma LPC composition at enrollment (A) and methyl-D₉-LPC and methyl-D₃-LPC enrichments (B) over the investigative period (4 days) for ARDS patients and controls. A significant difference in LPC composition was noted for LPC18:2 and LPC18:1. The LPC enrichment was calculated as percentage of total plasma LPC pool. Blue, methyl-D₉-LPC enrichment for patients; red, methyl-D₉-LPC enrichment for controls; green, methyl-D₃-LPC enrichment for patients; and purple, methyl-D₃-LPC enrichment for controls. Data are expressed as mean ± SEM. ¥P < 0.05.

Fig. 10.

Molecular specificity of fractional methyl-D₉-LPC (A, B) and methyl-D₃-LPC (C, D) enrichment for individual LPC species. Results are presented as the labeled fraction in the total plasma LPC pool for that individual PC species, compared between patient and control groups over the investigative period of 4 days. Each line color represents individual LPC species; blue, LPC16:0; red, LPC18:2; green, LPC18:1; purple, LPC18:0; yellow, LPC20:4; and black, LPC22:6. Data are expressed as mean ± SEM.