Mass Spectrometric Analysis of Surfactant Metabolism in Human Volunteers Using Deuteriated Choline

Abstract

Surfactant reduces surface tension at pulmonary air-liquid interfaces. Although its major component is dipalmitoyl-phosphatidylcholine (PC16:0/16:0), other PC species, principally palmitoylmyristoyl-PC, palmitoylpalmitoleoyl-PC, and palmitoyloleoyl-PC, are integral components of surfactant. The composition and metabolism of PC species depend on pulmonary development, respiratory rate, and pathologic alterations, which have largely been investigated in animals using radiolabeled precursors. Recent advances in mass spectrometry and availability of precursors carrying stable isotopes make metabolic experiments in human subjects ethically feasible. We introduce a technique to quantify surfactant PC synthesis in vivo using deuteriated choline coupled with electrospray ionization tandem mass spectrometry. Endogenous PC from induced sputa of healthy volunteers comprised 54.0 +/- 1.5% PC16:0/16:0, 9.7 +/- 0.7% palmitoylmyristoyl-PC, 10.0 +/- 1.0% palmitoylpalmitoleoyl-PC, and 13.1 +/- 0.3% palmitoyloleoyl-PC. Infusion of deuteriated choline chloride (3.6 mg/kg body weight) over 3 hours resulted in linear incorporation into PC over 30 hours. After a plateau of 0.61 +/- 0.04% labeled PC between 30 and 48 hours, incorporation decreased to 0.30 +/- 0.02% within 7 days. Compared with native PC, fractional label was initially lower for PC16:0/16:0 (31.9 +/- 8.3%) but was higher for palmitoyloleoyl-PC (21.0 +/- 1.2%), and equilibrium was achieved after only 48 hours. We conclude that infusion of deuteriated choline and electrospray ionization tandem mass spectrometry is useful to investigate surfactant metabolism in humans in vivo.

Phosphatidylcholine kinetics in neonatal rat lungs and the effects of rhuKGF and betamethasone

“…2). This is consistent with data on newly synthesized PC in adult mouse, rat, and human lung secretions, showing that it initially contributes a lower fraction to surf-PC and that molecular refinement occurs during surfactant recycling (5,8,17). However, whereas in adult organisms such refinement requires up to 24 h, our data show that in neonatal rat lungs refinement occurs within 3 h, indicating a much faster turnover, which is not affected by rhuKGF, betamethasone, or their combination.…”

“…D 4-choline was used as the internal standard, and calibration curves for individual components as external standards were used as previously described (8,9). In brief, for analysis, 100 l of upper phase was diluted with 1 ml of water.…”

“…Exchange of unsaturated fatty acids in position 2 via deacylation/reacylation (Lands cycle) results in the formation of additional PC16:0/16:0 but also contributes to PC16:0/14:0 synthesis (23,26). Here, the use of N-methyl-deuterated choline ([D 9 -methyl]choline), together with tandem mass spectrometry, offers the opportunity to analyze synthesis and turnover of PC molecular species, as well as of PC pools, in lung tissue and lung lavage fluid (LLF) (8,36).…”

Plasma Phosphatidylcholine Alterations in Cystic Fibrosis Patients: Impaired Metabolism and Correlation with Lung Function and Inflammation

“…Endogenous (unlabeled) choline and its derivative betaine as well as [methyl-D 9 ]choline, [methyl-D 9 ]betaine and the internal standard [1,1,2,2-D 4 ]choline were analyzed with electrospray ionization tandem mass spectrometry (ESI-MS/MS) [24], using an Atlantis® HILIC Silica 3µm column (2.1x30mm; 50°C) using a HP1100 LC system (Agilent Technologies, Heilbronn, Germany) on a Waters Micromass Quattro Micro API triple quadrupole mass spectrometer (Waters, Dreieich, Germany), supplied with an electrospray ionization interface. Components were analyzed by specific reaction monitoring (SRM) in the positive ionization mode, using mass by charge (m/z) transitions of 104 60 (choline), 108→61 (D 4 -choline), 113→69 ([D 9 -methyl]-choline), 118→59 (betaine), 127→68 ([D 9 -methyl]-betaine), 184→86 (phosphocholine) and 193→95 (D 9 -phosphocholine).…”

Specificity and rate of human and mouse liver and plasma phosphatidylcholine synthesis analyzed in vivo

“…We previously applied ESI-MS/MS methodology to quantify the synthesis and turnover of individual molecular species of PC in lung surfactant in human volunteers ( 20 ) and now use it to describe the synthesis of individual molecular species of hepatic PC in vivo. Initial analyses were conducted in a mouse model to establish whether analysis of the molecular specifi city and dynamics of label incorporation into plasma PC can provide a direct insight into liver PC metabolism.…”