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

Department of Neonatology, Faculty of Medicine, Eberhard-Karls-University, Tübingen; Department of Biochemistry, NM Institute of the Eberhard-Karls-University, Reutlingen; Departments of Pediatric Pulmonology and Neonatology and Respiratory Medicine, Hannover Medical School, Hannover, Germany; and Division of Infection, Inflammation and Repair, School of Medicine, Southampton General Hospital, Southampton, United Kingdom

Correspondence and requests for reprints should be addressed to Wolfgang Bernhard, M.D., Ph.D., Department of Neonatology, Faculty of Medicine, Eberhard-Karls-University, Calwer Straße 7, D-72076 Tübingen, Germany. E-mail: wolfgang.bernhard{at}med.uni-tuebingen.de

	ABSTRACT

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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.

Key Words: human surfactant metabolism • induced sputum • stable isotopes • mass spectrometry • phosphatidylcholine molecular species

Pulmonary surfactant is a complex mixture of phospholipids, neutral lipids, and specific proteins. It is essential for normal lung function by reducing surface tension at the air–liquid interface of terminal air spaces (1–3). Phospholipids comprise 80% of surfactant, of which 80–85% are phosphatidylcholine (PC) molecular species (1, 4). Among these PC species, dipalmitoyl–PC (PC16:0/16:0), the principal surface tension-lowering molecule, comprises 40–60%, with palmitoylmyristoyl–PC and monounsaturated palmitoylpalmitoleoyl–PC and palmitoyloleoyl–PC contributing a further 30–40% of total PC (4–6). We previously demonstrated a large variability of PC16:0/16:0, palmitoylmyristoyl–PC, and palmitoylpalmitoleoyl–PC that correlates with differences in respiratory physiology (5, 7, 8). Other studies demonstrated that surfactant PC composition is altered in response to pulmonary injury (9–11). Metabolism of surfactant PC, however, has mostly been investigated in animal models because the injurious effects of radioactively labeled precursors preclude application in humans. Recently, labeling of glucose and fatty acids with the stable isotope ¹³C, combined with combustion interface isotope ratio mass spectrometry, has been introduced for the investigation of surfactant metabolism in preterm infants (12–14). Moreover, exogenous surfactant labeled with stable isotopes has been used to monitor surfactant kinetics in infants (13). However, large amounts of labeled precursors, for instance, 245 mg/kg of ¹³C-labeled glucose, have been used; subsequent analyses were time consuming and did not reveal metabolism of individual PC species (12–14). We have previously demonstrated that incorporation of choline labeled at its methyl groups with the stable isotope deuterium ([methyl-D₉]choline) followed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) provides a sensitive approach to quantify the specificity of PC synthesis in cultured cells (15, 16). In this study, we describe a method to monitor surfactant PC metabolism in humans using [methyl-D₉]choline combined with ESI-MS/MS analysis of endogenous and [methyl-D₉]choline-labeled individual PC species from induced sputa. Our aim was to establish and validate this technique in healthy human volunteers. For comparison with the lungs, kinetics of [methyl-D₉]choline incorporation into plasma PC, which is a reflection of liver synthesis and secretion, was investigated. This is the first study to demonstrate feasibility of metabolic analysis of surfactant PC molecular species in humans in vivo using [methyl-D₉]choline together with ESI-MS/MS. Some of the results of this study have previously been reported in the form of an abstract (17, 18).

	METHODS

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Volunteers and Study Design
Three female volunteers and one male healthy volunteer (22 to 27 years, 63 to 92 kg body weight; Table 1) with normal physical activity participated in the study after approval by the local ethics committee at Hannover Medical School, written consent, and clinical examination by an independent physician. Volunteers had no history of lung disease and had normal values for FEV₁ and PEF obtained by spirometry according to American Thoracic Society recommendations and were nonsmokers. No participant displayed clinical or electrocardiographic signs of cardiac disease. Standard laboratory parameters showed no signs of impaired renal or hepatic function. At the screening visit, volunteers underwent a test protocol of sputum induction, where feasibility and safety was proved by spirometry. [Methyl-D₉]choline chloride (3.6 mg/kg body weight) was infused intravenously over a period of 3 hours. Ethylenediaminetetraacetic acid blood was collected from 1–168 hours after the start of infusion, centrifuged at 1000 x g for 15 minutes, and plasma aspirated. Induced sputa were collected in four intervals of 5 minutes at each time point before (t = 0 hours) and from 12–168 hours after the start of infusion. Sputum cells were sedimented by centrifugation at 400 x g for 10 minutes. Plasma and sputa were then stored at –20°C, and cytospins of sputum cells were stained with May-Grünwald-Giemsa and differentiated (see online supplement).

Statistical Analysis
Data are expressed as mean ± SE. To compare groups, two-tailed t tests were used, and in the case of three or more groups, analysis of variance was performed with the Bonferoni correction method for multiple group comparisons using GraphPad Instat Version 3.05 (GraphPad Software, San Diego, CA). Linear regression analyses were performed using the same software.

	RESULTS

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Volunteers completed the study without developing airway symptoms as assessed by peak flow measurements before and after sputum induction throughout the study. Sputum volumes ranged from 1 to 4 ml per portion. Epithelial cells of sputa comprised 88.3 ± 1.3%. Among the leukocytes, the fourth sputum fraction contained more macrophages than the first fraction (75.0 ± 3.0% [n = 34] vs. 45.3 ± 4.4% [n = 35], respectively; p < 0.0001) at the expense of neutrophils. Lymphocytes and eosinophils were generally below 2% (see Table E1 in the online supplement).

Composition of Phospholipid Molecular Species in Sputum
Native sputum PC consisted of 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 as major components. Less abundant PC components detected were the alkylacyl ether species palmitylpalmitoyl–PC (3.2 ± 0.3%) and palmitylstearoyl–PC (1.8 ± 0.1%) and the polyunsaturated species palmitoyllinoleoyl–PC (6.9 ± 0.2%) and palmitoylarachidonoyl–PC (1.4 ± 0.1%). The composition of native PC species was different in the first compared with subsequent sputum fractions, with increased concentrations of palmitoyloleoyl–PC, palmitoyllinoleoyl–PC, and palmitoylarachidonoyl–PC (see Figures E1 and E2 and Tables E3 and E4 in the online supplement).

Kinetics of [Methyl-D₉]choline in Blood Plasma
Concentrations of endogenous choline in blood plasma did not change during or after infusion of [methyl-D₉]choline chloride (8.4 ± 0.3 µM). During infusion, mean values of [methyl-D₉]choline were 8.2 ± 0.9 µM but decreased to near zero within 3 hours after end of infusion (see Figure E3 in the online supplement).

Incorporation of [Methyl-D₉]choline into Sputum PC
As demonstrated in Figure 1A , incorporation of D₉ label into PC increased until 30 hours after the start of [methyl-D₉]choline infusion. Incorporation was linear from 12–30 hours (r = 0.9998, p < 0.05), and extrapolation of this regression line to the x axis intercept suggested a lag phase of 5.7 hours before labeled PC appeared in sputa. A period of constant labeling was achieved from 30–48 hours (0.61 ± 0.04% [methyl-D₉]choline-labeled PC), after which the enrichment of [methyl-D₉]choline in PC decreased by 50% within 5 days (0.30 ± 0.02%, p < 0.001).

View larger version (30K): [in this window] [in a new window]   Figure 1. Time course of [methyl-D9]choline incorporation into total phosphatidylcholine (PC) from induced sputa and blood plasma. Data indicate the fraction of D9-choline–labeled PC relative to native PC of (A) sputa from four volunteers at 12 to 168 hours and of (B) blood plasma of the same individuals from 1 to 168 hours after start of 3 hours of infusion of 3.6 mg/kg body weight [methyl-D9]choline chloride. Data are means ± SE. (A) p < 0.01 vs. T = 12 hours; p < 0.001 vs. t = 12 hours; p < 0.05 vs. t = 48 hours; ¶p < 0.001 vs. t = 48 hours. (B) *p < 0.05; p < 0.001 vs. baseline (0–2 hours); p < 0.001 vs. 6 hours; ||p < 0.001 vs. 24 hours.

View larger version (26K): [in this window] [in a new window]   Figure 2. Time course of fractional [methyl-D9]choline incorporation into individual PC species of sputa. Fractional [methyl-D9]choline incorporation into individual PC species, in relation to [methyl-D9]choline label of total PC, was calculated from total parents of m/z = 193 at the given time points. Data are means ± SE of four sputa from four volunteers at each time point. p < 0.01; p < 0.001 vs. t = 24 hours; p < 0.001 vs. equilibrium (48 to 168 hours).

	DISCUSSION

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Developmental and Pathologic Impact of Surfactant PC Species
Phospholipids comprise the major components of lung surfactant, and variability of their molecular composition correlates with differences in respiratory physiology (7, 8, 21). Additionally, differences in surfactant phospholipid molecular species composition have been described in a variety of lung diseases, including neonatal and acute respiratory distress syndrome, pneumonia, cystic fibrosis, and asthma (1–3, 9–11); however, the extent to which such changes are due to alterations in either synthesis or hydrolysis of phospholipids has not been established. Such analysis requires the characterization of phospholipid metabolism in humans in vivo, but one major restriction has been the harmful effects of radioactively labeled substrates. Methods for analysis of phospholipid synthesis that monitor incorporations of nonradioactive stable isotopes offer obvious advantages for human studies. Previous approaches to analysis of surfactant PC metabolism have used glucose or fatty acid precursors as well as exogenous surfactant labeled with the carbon isotope ¹³C and have then analyzed ¹³C enrichment of phospholipids by combustion interface isotope ratio mass spectrometry (12–14). Such analyses, however, provide minimal information about metabolism of individual PC molecular species. Using the combination of [methyl-D₉]choline chloride incorporation together with ESI-MS/MS described here, investigation of human lung surfactant metabolism is feasible using as little as 3.6 mg of [methyl-D₉]choline chloride per kg body weight, and results are generated in terms of individual PC molecular species. Additionally, in contrast to the extensive derivatization steps required for combustion interface isotope ratio mass spectrometry– or HPLC-based approaches (8, 12–14), phospholipid extracts did not have to be fractionated or derivatized before ESI-MS/MS analysis (6).

PC16:0/16:0 is synthesized both directly de novo from diacylglycerol and by acyl remodeling exchange of the fatty acid at the sn-2 position of unsaturated PC (reviewed in 22). Radioactively labeled choline and palmitate have been used to investigate PC metabolism in animal models. Analysis of palmitate incorporation in vivo is complex as it can originate from the circulation as well as from synthesis within the lungs, is incorporated into various lipids, metabolized to unsaturated palmitoleic acid (23), and targeted to ß-oxidation. In contrast, choline is essentially derived from the circulation, and only negligible amounts are incorporated into other lipids, such as sphingomyelin. Initial choline incorporation into total PC and its molecular species reflects PC synthesis de novo, whereas subsequent changes to the incorporation pattern of PC molecular species reflect the activity of subsequent acyl remodeling (22). The study presented here is the first to describe surfactant PC metabolism in humans using deuterium-labeled choline ([methyl-D₉]choline) combined with subsequent ESI-MS/MS analysis of the kinetics of precursor incorporation into total PC and its major molecular species. The shift of individual components and their product ion by 9 mass units led to excellent separation of native and labeled PC species (for details, see the attachment in Table E2 in the online supplement).

Labeling of Human Surfactant and Blood Plasma PC with [Methyl-D₉]choline
Sputum samples demonstrated a nearly linear increase of [methyl-D₉]choline incorporation from 12–30 hours after the start of infusion in sputa and until 24 hours in plasma PC. Assuming that the plateau of sputum PC label (0.61 ± 0.04%) represents maximal alveolar label and that the alveolar pool size of PC is 266 ± 28 µmol (calculated from saturated PC) (24), the maximum concentration of deuteriated PC within the alveolus is 1.62 ± 0.11 µmol, which amounts to 0.1% of the administered dose (250 mg of [methyl-D₉]choline chloride per 70 kg body weight = 1.68 mmol). Similarly, assuming a plasma volume of 3l and a plasma PC concentration of 1.6 ± 0.1 mM (25, 26) gives a value of 4.8-mmol plasma PC, of which 0.45 ± 0.06% (21.6 ± 2.9 µmol) are D₉-labeled plasma PC, equivalent to 1.3 ± 0.2% of the administered dose. Consequently, only trace amounts of [methyl-D₉]choline were incorporated into both surfactant and plasma PC.

Although bronchoalveolar lavage (BAL) fluid is a common source for studying surfactant composition as well as kinetic aspects of alveolar metabolism, we used induced sputum because repetitively harvesting induced sputum is less invasive than BAL. Several studies have demonstrated that in healthy lungs airway phospholipids are representative of those in the alveoli, as the airways do not secrete significant amounts of phospholipid (4, 26, 27). The data presented here are in agreement with those studies and show that induced sputum displays an overall phospholipid composition identical to that of BAL fluid (4, 6). Increases in unsaturated PC species at the expense of PC16:0/16:0 and palmitoylmyristoyl–PC in the first sputum fractions, albeit significant, were negligible, as they always were in a range of less than 5%. Comparability of sputum phospholipids with those from BAL fluid was also confirmed by exemplary analysis of anionic phospholipids (see Figure E2 and reference 6 in the online supplement). Consequently, we state that the use of induced sputum in this study is compatible with our aim of measuring surfactant PC metabolism. Nevertheless, because of the mucociliary transit time, sputum samples do not correctly reflect surfactant kinetics of the alveoli. Moreover, contaminations by phospholipids from airway epithelia and from invaded inflammatory cells may be relevant under pathologic conditions (4, 26). Therefore, using BAL fluid may be preferable in future studies on patients.

The kinetics of alveolar surfactant metabolism analyzed from induced sputa are influenced by the uptake of choline from the circulation, kinetics of precursor processing within the type II pneumocytes, secretion of surfactant into the alveolar spaces, alveolar turnover, and the mucociliary transit time from lung periphery to central airways. It is known that at choline dosages of up to 0.2 mmol/kg body weight (instead of 24 µmol/kg as used in this study), infused choline is rapidly removed from the circulation via uptake into tissues (28, 29). In mice, maximal label of lung tissue occurs within 1.5 hours after injection of [methyl-³H]choline, followed by a plateau of at least 24 hours (30). Our data on the rapid removal of [methyl-D₉]choline from the circulation are consistent with these findings. They suggest loading of tissues with [methyl-³H]choline during and shortly after end of infusion, whereas the time course of PC labeling in lungs and plasma results from subsequent processes of synthesis, intracellular trafficking, secretion, and turnover.

Similar to experiments in mice (30), [methyl-D₉]choline incorporation into sputum PC was linear over a range of 18 hours (0.025% increase of label per hour). The x-intercept of the regression curve (5.7 hours after start of infusion) represents the delay of sputum PC labeling, caused by a combination of the kinetics of [methyl-D₉]choline uptake into type II pneumocytes, PC synthesis, lamellar body packaging and surfactant secretion, and the mucociliary transit time of surfactant from the alveoli to those airspaces where it was accessed by sputum induction. The time from uptake of [methyl-D₉]choline from the circulation until appearance of labeled PC in the alveoli lasts not more than 1.5 hours in animal experiments (30, 31). Similarly, for labeled plasma PC, mostly originating from the liver (32), the x-intercept was only 1.6 hours in this study. These estimates indicate that the proportion of the time delay for appearance of labeled PC in sputum that could be attributed to processes within the type II pneumocyte was probably in the order of 1.5 to 2 hours. Consequently, the proportion of the delay caused by movement of labeled surfactant from the alveolus to more central airways can be estimated at the remaining 3.5 to 4 hours. In contrast, the plateau from 30–48 hours and the slow subsequent decrease of label (50% in 7 days) represent the time course of storage and recycling of surfactant PC in healthy volunteers with normal physical activity.

Metabolism of [Methyl-D₉]choline-labeled PC Molecular Species
Although composition of native PC species in sputum was representative of alveolar surfactant, this was not the case for [methyl-D₉]choline-labeled PC species after 12 hours, and equilibrium with the endogenous PC pool was achieved after only 48 hours. Estimates of the relative contributions of synthesis de novo and fatty acyl remodeling suggest that both contribute approximately 50% to the synthesis of PC16:0/16:0 (33). It is further assumed that this process essentially takes place before surfactant storage in lamellar bodies and secretion into the alveolar spaces (22, 34). However, the data presented here demonstrate that the fractional label of PC16:0/16:0 initially only comprised 30% instead of more than 50% after reaching the equilibrium, whereas labeling of unsaturated PC species was increased. Similarly, in mice, the fractional label of PC16:0/16:0 in BAL fluid initially is only 30%, whereas it takes 24 hours until it is in equilibrium with native alveolar PC16:0/16:0 (27). Although we have no unambiguous explanation of these findings, they question the accepted paradigm of de novo synthesis and acyl remodeling, followed by storage and subsequent secretion of "mature" surfactant. Instead, they suggest that as material reflecting the pattern of synthesis de novo is secreted, a substantial portion of newly synthesized PC is rapidly incorporated into lamellar bodies and secreted before it has time to become remodeled to equilibrium surfactant composition.

Conclusions
We conclude that in vivo labeling with [methyl-D₉]choline combined with ESI-MS/MS analysis of phospholipids is a feasible and safe technique to study human surfactant PC metabolism. Depending on physiologic or clinical questions, samples may be harvested via sputum induction or BAL of patients. Analysis of labeling of PC molecular species reveals that a metabolic short cut may exist that leads to the secretion of a PC fraction that has not undergone the complete sequence of de novo synthesis followed by acyl remodeling.

	Acknowledgments

 
The authors thankfully acknowledge the excellent technical assistance of Mrs. Christa Acevedo and Ms. Marion Schaël.

	FOOTNOTES

 
Supported by Nycomed Pharma GmbH, Unterschleißheim, Germany, by an institutional grant of the Medical Faculty of the University of Tübingen (F.1275089) and by grants 055490 and 457405 from the Wellcome Trust.

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Conflict of Interest Statement: W.B. received 5,840.80 DM, which is approximately $3,650, from Nycomed GmbH, FRG, in 2001 as support for conducting this study; C.J.P. was supported by a studentship sponsored by Britannia Pharmaceuticals, characterization of a therapeutic surfactant 2001–2004 £57,055; A.J. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; G.A.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; J.M.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; J.F. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; C.F.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; D.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; A.D.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this article.

Received in original form January 20, 2004; accepted in final form March 18, 2004

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