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

Background: Liver impairment, ranging from steatosis to cirrhosis, is frequent in cystic fibrosis (CF) patients and is becoming increasingly significant due to their improved life expectancy. One aspect of hepatic alterations is caused by increased fecal loss of the essential nutrient choline, following enterohepatic bile phosphatidylcholine (PC) cycle impairment. Hepatic PC synthesis, both de novo and via phosphatidylethanolamine-N-methyl-transferase (PEMT), is essential for very low-density lipoprotein (VLDL) secretion. VLDL-PC in particular contributes to the organism's supply with polyunsaturated fatty acids (LC-PUFA), namely arachidonic (C20:4) and docosahexaenoic acid (C22:6). Consequently, choline deprivation and altered hepatic PC metabolism may affect plasma PC homeostasis and extrahepatic organ function. Objectives: To investigate relationships between altered plasma choline and PC homeostasis and markers of lung function and inflammation in CF. To assess alterations in hepatic choline and PC metabolism of CF patients. Design: Quantification of plasma/serum choline and PC species in adult CF patients compared to controls. Correlation of PC with forced expiratory vital capacity (FEV1) and interleukin 6 (IL-6) concentrations. Analysis of choline and PC metabolism in CF compared to controls, using deuterated choline ([D9-methyl]-choline) labeling in vivo. Results: Mean choline and PC concentrations in CF patients were lower than in controls. Choline and PC concentrations as well as fractions of C22:6-PC and C20:4-PC correlated directly with FEV1, but inversely with IL-6. Plasma concentrations of deuterated PC were decreased for both pathways, whereas only in PC synthesized via PEMT precursor enrichment was decreased. Conclusion: In CF patients, hepatic and plasma homeostasis of choline and PC correlate with lung function and inflammation. Impaired hepatic PC metabolism, exemplarily shown in three CF patients, provides an explanation for such correlations. Larger studies are required to understand the link between hepatic PC metabolism and overall clinical performance of CF patients, and the perspective of choline substitution of these patients.


Introduction
Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), a cyclic adenosine monophosphate activated chloride channel [1]. Improved treatment of exocrine pancreas insufficiency by enzyme substitution and inhibition of pulmonary decay by antibiotic, enzymatic and physical therapies have significantly improved life expectancy within the last 30 years, resulting in median survival between 38.3 and 48.5 years [2,3]. As a result of such improvements, liver disease has started to become a clinical issue in CF. It is assumed that hepatobiliary abnormalities occur in approximately 13-27% of pediatric CF patients and 30-72% of adults with CF, although being frequently subclinical. Alterations range from hepatic steatosis and gallstones to cholestasis, cholangitis, fibrosis and focal biliary cirrhosis [4][5][6][7]. Minor affections as hepatosteatosis or gallstones should be discriminated from CF related liver disease (CFRLD), which frequently leads to fibrosis and cirrhosis. The former are primarily related to malnutrition and nutrient losses, like choline, linoleic acid and other long chain polyunsaturated acids (LC-PUFA), where the enterohepatic choline metabolism is a potential key factor [4,8,9]. Animal experiments show that impaired choline, phosphatidylcholine (PC) and essential fatty acid homeostasis is linked to liver failure [4,10,11]. However, these nutrients are relevant beyond liver function (see below).
A link from the genetic defect to liver impairment in CF is the loss of CFTR function in cholangiocytes [12]. Here, impaired chloride secretion affect bicarbonate, electrolyte, fluid and pH homeostasis. In the Phe508del mouse model of CF, cholangiocyte impairment is associated with an increased secretion and fecal losses of bile salts. Additionally, biliary phospholipid secretion and fecal losses are increased [7]. In human CF patients, fecal losses of choline metabolites derived from biliary phosphatidylcholine (PC), are increased despite pancreatic enzyme replacement. Fecal losses of undigested PC are 9-fold increased, suggesting that impaired pancreatic phospholipase A 2 (pPLaseA 2 ) function is causative here, and choline deficiency may be central to metabolic alterations in CF [13][14][15].
The clinical relevance of the enterohepatic PC/choline cycle for CF bases on quantitative as well as qualitative aspects: a 1500g adult liver comprises about 30mmol (22.5g) PC, of which 5-10g/d are secreted via bile [16,17]. This equals a daily turnover of 22-44% of total liver PC, being equivalent to 0.7-1.4g choline, a 1.25-2.5 fold of the adequate daily intake (AI) of an adult male (0.55g/d) [18]. Characteristically, maintenance of biliary PC secretion has biological priority over liver integrity and choline/PC distribution to peripheral organs via lipoproteins [11]. Sufficient hepatic PC synthesis using choline, or via methylation of phosphatidylethanolamine (PE) by PE-N-methyltransferase (PEMT), are essential to the assembly and secretion of very low density lipoproteins (VLDL), thereby integrating hepatic lipid and precursor metabolism with peripheral organ supply, since VLDL comprise about 20% PC [19]. Moreover, choline is a source to generate betaine, a methyl-group donator to regenerate methionine from homocysteine, the former being essential for the PEMT pathway [20,21]: the activated form of methionine, S-adenosyl-methionine (SAM), is co-substrate of PEMT for PC formation from polyunsaturated PE. Here, deficiency in methyl donators is consistent with decreased methionine and increased homocysteine concentrations in CF children [22,23]. Hence, fecal choline losses in CF may affect choline, one-carbon unit, PC and LC-PUFA homeostasis of the liver. Due to the central role of this organ, these considerations suggest an impairment of peripheral supply and functional consequences,

Materials and Methods
The study was approved by the local Ethics committee (University of Tübingen) and written and informed consent obtained by participants

Chemicals
Chloroform was of HPLC grade and from Baker (Deventer, The Netherlands). Methanol, water and ammonium hydroxide were of analytical grade and from Fluka Analytical/Sigma-Aldrich (Munich, Germany). Phospholipid standards were from Avanti Polar Lipids (Alabaster, AL, USA) or from Sigma-Aldrich (Munich, Germany), and purity checked by tandem mass Spectrometry (see below). Deuterated choline ([D 9 -methyl] choline) chloride was purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). All other chemicals were of analytical grade and from various commercial sources.

Study population
Eleven CF patients (5 females; all homozygous for Phe508del and 10 volunteers (5male/5female) participated in the study. All CF patients had ambulatory control visits at least every 3 months, and no CF patient had a history of allergic bronchopulmonary aspergillosis requiring systemic glucocorticoid treatment. Patients received standard treatment with pancreatic enzyme replacement, inhaled glucocorticoids & beta-mimetics, and antibiotic treatment in case of Pseudomonas aeruginosa colonization (N=10). 3 male CF patients and 4 (1 female) healthy volunteers approved the [D 9 -methyl]choline labeling experiments. Of these latter CF patients, one showed no pathological ultrasound or serologic signs of liver disease, whereas one was diagnosed with steatosis, but no cirrhosis. The third patient suffered from liver cirrhosis as evident on ultrasound.

Lung function and IL-6 determination
Lung function of the CF patients was assessed during their visits to the local CF clinic from 2007-2009 (7-9 per patient) using bodyplethysmography/spirometry (Jaeger, Höchberg, Germany). Blood samples were also taken. IL-6 concentration was determined in serum by the Tuebingen university hospital clinical laboratory. A plasma aliquot was prepared from each sample, which was processed as described below.

Labeling of patients with [D 9 -methyl]choline
A sterile aqueous solution of [methyl-D 9 ]choline chloride (25mg/ml) was prepared by the Tuebingen university hospital's pharmacy department, aliquoted in 5 and 10ml glass vials, and stored at -80°C until use. Representative samples were analyzed to ensure that they were sterile and to confirm the absence of pyrogens. All procedures were performed under standardized conditions, according to good pharmaceutical practice, by the local pharmaceutical unit at the University of Tübingen, as described before. For infusion, aliquots of stock solutions equivalent to 3.6mg/kg body weight were drawn into a 50ml syringe and sterile isotonic saline added to make 50ml total volume. This solution was continuously infused into a forearm vein of overnight fasted participants over 3 hours (0.278ml/min) using an infusion pump. Patients were mobile and were allowed to drink water during the infusion. Eating was allowed following completion of High performance liquid chromatography of plasma PC Routine analysis of PC molecular species and sphingomyelin (SPH) was performed from lipid extracts using high performance liquid chromatography (HPLC) as described before [26]. Briefly, PC/SPH was prepared from 500nmol plasma phospholipid aliquots using 100mg Strata NH 2 disposable cartridges (Phenomenex Inc., USA) and dimyristoyl-PC as an internal standard. Isocratic separation of individual molecular species was then performed with a 4.6 x 250mm Sphere Image ODS II column (Schambeck, Bad Godesberg, Germany) at 50°C, methanol:water choline chloride (925:75:7.6, v/v/w) as the mobile phase and post-column fluorescence derivative formation of PC in the presence of 1,6-diphenyl-1,3,5-hexatriene [25].

ESI-MS/MS analysis of endogenous and stable isotope labeled PC and of endogenous PE
Analysis of phospholipids containing a choline headgroup (PC, lyso-PC, SPH) and of PE was performed as described before [21,27], using a Thermo TSQ Quantum Discovery MAX triple quadrupole mass spectrometer (Thermo, Dreieich, Germany) equipped with a heated electrospray ionization (H-ESI) interface. In brief, 25µL sample (6µmol/L phospholipid in butanol:methanol:water (75:23:2,v/v)) containing dieicosanoyl-PC and dimyristoyl-PC as internal standards were introduced via loop injection using a Finnigan Surveyor Autosampler Plus and MS Pump Plus (Thermo, Dreieich, Germany), and a mobile phase comprising butanol:methanol:water:25% NH 4 OH (75:23:1.7:0.3;v/v). PC species were quantified from precursor ions of the respective diagnostic fragment ions. These were phosphocholine for endogenous PC, lyso-PC and SPH (mass / charge (m/z)=184), D 9 -phosphocholine (m/z=193) for PC synthesized de novo by direct [methyl-D 9 ]choline incorporation, and D 3 -phosphocholine (m/z=187) and D 6 -phosphocholine (m/ z=190) for PC synthesized by sequential PE N-methylation to yield PC. Here, sequential transfer of one or two deuterated methyl groups (CD 3 -) from D 3 -S-adenosylmethionine originating from [D 9 -methyl]-choline catabolism leads to mass shifts of PC species of M+3 and M+6. PE species were analyzed from the neutral loss fragment phosphoethanolamine (m/z=+141) of the individual masses.

Calculation of MS/MS data
Total ion counts of PC and PE species analysis were collected in Excel spread sheets using XCalibur® Version 2 Software (Thermo). Data were then processed using an Excel-based in-house macro for correction of 13 C isotope effects of individual molecular species and reduced response with increasing m/z values. According to the low enrichment of PC with D 3 -methyl groups via PEMT, (M+3; m/z=187), overspill from the diagnostic fragment of the naturally occurring M+3 analogue (m/z=187) of endogenous PC was included in the calculation of D 3 -labelled PC. From these data the molecular composition of endogenous PC species and those newly synthesized via de novo synthesis (D 9 -labeled PC) as well as via PE methylation pathway (D 3 -+D 6 -labeled PC) were calculated. Concentrations of individual PC species were calculated from the internal standard. From these data, we determined the D 9 -, D 3 -& D 6 -enrichments and their absolute plasma concentrations at time points (3 to 72h after start of infusion) for total PC, individual PC species and PC subgroups. The D 3 -and D 6 -enrichment of PC was used to calculate the D 3 -enrichment of SAM, according to the formula (ΣD6-PC/(ΣD3-PC+ΣD6-PC)) as described before [21]. PC and PE subgroups were generated from individual PC molecular species by summing up species containing a monounsaturated (oleic acid, C18:1), diunsaturated (linoleic acid, C18:2), arachidonic (C20:4) or docosahexaenoic (C22:6) acid [27].

Statistical analysis
Data are indicated as means and standard deviation. Statistical analysis was performed using Instat, version 3.10 (GraphPad, La Jolla, CA, USA). Spearman Rank test was used for regression analysis after test for linearity, and the Mann-Whitney-test for group comparisons. P values below 5% were regarded as statistically significant.

Endogenous choline, betaine and phosphatidylcholine concentrations
In CF patients plasma choline concentration was 8.51±0.61 µmol/L (mean±SE), whereas in control subjects it was 10.85±0.96 µmol/L (p<0.05). In those individuals participating in the [D 9 -methyl]choline labeling experiment endogenous choline concentration did not change over the whole sampling period (N=10 samples over 72h), and was 6.40±0.32 and 7.82±0.27µmol/L in CF and control subjects, respectively (p<0.01). Endogenous betaine was 20.88±2.09µmol/L for the whole CF collective, compared to 30.28±4.15µmol/L in controls (p<0.05).

Correlation of plasma choline and PC concentration with lung function
Concentrations of choline as well as of total PC directly correlated with FEV 1 (Fig. 2A, B). By contrast, IL-6 as an inflammation marker was inversely related to choline and PC (Fig. 2C,  D). We then set out to investigate whether there was any relationship between the fractions  of PC sub-groups and the clinical parameters measured. Fractions of C20:4-PC and C22:6-PC were increased in patients with better lung function (FEV 1 ) (Fig. 3A, B). By contrast, they were inversely related to IL-6 concentrations in serum (Fig. 3C, D). However, these overall correlations between choline and total PC concentrations and FEV 1 or IL-6 values were not consistently found intra-individually. Here correlation coefficients showed a wide range for choline and total PC concentrations, particularly relative to FEV 1 ( Fig. 2A, B). This similarly applied to the correlations between the C20:4-PC and C22:6-PC fractions versus FEV 1 (Fig.  3A, B). In particular, the correlation of FEV 1 versus C22:6-PC (Fig. 3B), but also FEV 1 versus choline, total PC and C20:4-PC ( Fig. 2A, B, Fig. 3A) suggested the existence of two patient subgroups. By contrast, there was no significant correlation between plasma betaine and FEV 1 or IL-6 (data not shown).

Concentrations and deuterium-enrichment of water soluble PC precursors
We then investigated, whether in CF patients compared to control subjects choline and PC metabolism is characteristically different. Detailed patient and control data are given in Table 1. While body size and weight in these CF patients were below those of the controls, their body mass indexes were almost identical. Clinical data showed no major impairments in terms of blood protein and cells, coagulation, biliary function, hepatocyte integrity (AST, ALT, LDH, AP and γGT), and renal function. FEV 1 was lower in CF patients compared to controls. As expected, blood leucocytes were higher in CF patients, whereas serum cholesterol was lower.
There was no difference in the plasma kinetics of [D 9 -methyl]-choline between CF patients and controls (Fig. 4A). [D 9 -methyl]-choline rapidly increased  -methyl]choline chloride (250mg/70 kg body weight) was administered over a 3h period to fasting individuals. Plasma samples were drawn at t=1h, 2h, 3h, 6h, 9h, 24h, 33h, 48h and 72h and analyzed by electrospray tandem mass spectrometry. Endogenous and D 9 -labeled choline and betaine were determined in plasma, and D 9 -enrichment of choline (A) and Betaine (B) are indicated as the ratio of the respective D 9 label compared to endogenous metabolite. D 3 -enrichment of SAM over time (C) was calculated from the D 6 -/(D 3 +D 6 ) ratio of the respective metabolites as described elsewhere [21]. Data are means ± SD of 4 controls (open symbols) and 3 CF patients (filled symbols).    -methyl]-enrichment in betaine produced no plateau, and maximum enrichment in CF and controls was achieved at the end of infusion (3h) (17.8±2.7 and 20.3±3.5%, respectively (p<0.05). D 9 -methyl]-betaine persisted for longer in the circulation in both CF patients and controls, and enrichment after 24 hours after onset of the infusion was 1.16±0.17% and 1.32±0.47%, respectively (p>0.05). Endogenous phosphocholine was at the limit of detection, and its deuterated derivative ([D 9 -methyl]phosphocholine) below detection limit in both groups (data not shown). The D 3 -enrichment of methionine as calculated from D 3 -and D 6 -labeled PC [21] was detectable from the end of [D 9 -methyl]choline infusion (3h), and showed no significant difference between CF patients and controls (Fig. 4C). Mean values of enrichment were 1.30±0.30% and 1.15±0.37% at 3-6h, respectively (p>0.05), and then decreased to 0.40±0.05% and 0.43±0.08% from 9-72h, respectively (p>0.05).

Precursor enrichment and concentrations of D 9 -and D 3/6 -labeled plasma PC
[D 9 -methyl]choline enrichment of PC approached a maximum of 0.535±0.061 and 0.561±0.038% at 33h in controls and CF, respectively (p>0.05) (Fig. 7A). D 3 /D 6 -enrichment of plasma PC representing the PEMT pathway (Fig. 7B) peaked at 0.226±0.016% in controls (which is 29.7±2.1% of total enrichment) 33h following the onset of the infusion. In CF patients, however, values were lower, with a maximum of 0.174±0.011 (p<0.01), which is only 23.7±1.5% of total enrichment. From these enrichments and total (endogenous and deuterated) PC concentrations, we calculated the concentrations of D 9 -and D 3 /D 6 -labeled PC in plasma of CF patients vs. controls. Concentration of D 9 -labeled PC linearly increased from 3 to 9h after start of infusion, with increments of 0.880µmol/L*h -1 (R 2 =0.998) and 0.535µmol/  L*h -1 (R 2 =0.996) for controls and CF patients, respectively. The curve than flattened, and concentration differences were significant from 24h after start of infusion onwards, with a concentration of D 9 -labeled PC in CF comprising 62.4±4.3% of controls (p<0.001) (Fig.  7C). The increment in D 3/6 -labeled PC from 3 to 9h was 0.162µmol/L*h -1 (R 2 =0.873) and 0.101µmol/L*h -1 (R 2 =0.963) for controls and CF patients respectively. Concentrations in CF between 24 and 72h were significantly lower than in controls (Fig. 7D), comprising 46.9±2.6% of controls, which was a stronger decrease than that of D 9 -PC (p<0.01).
LC-PUFA metabolism is intimately linked to that of choline, since PC is a major LC-PUFA carrier between the liver and other organs. Moreover, plasma PC is an important marker of an organism's LC-PUFA status, whereas cholesterol esters and triglycerides are of minor importance here [21,27,31,32]. Consequently, choline and PC homeostasis not only impact on liver metabolism, but also affect extrahepatic, clinically important parameters in CF: the lungs are compromised by chronic inflammation, even in the absence of bacterial or viral infection [33]. The release of IL-6 induces the formation of acute phase proteins by the liver, and is involved in the cross-talk between organs during inflammatory processes including lungs and liver [34][35][36]. The C20:4 and C22:6 moieties of plasma PC are important precursors of prostaglandins, leucotrienes and resolvins, being involved in inflammation and organ integrity [37]. Choline, an essential nutrient to all cells for membrane PC formation, particularly during cell proliferation and epithelial repair, and for the synthesis of VLDL-PC in liver, is deficient in CF patients [13,18]. Consequently, clinically important characteristics in CF disease, like impaired lung function and chronic inflammation, may be linked to impaired homeostasis, hepatic metabolism and peripheral supply of choline, total PC and LC-PUFA-PC. Interestingly, pro-apoptotic ceramides are increased in CF lungs [38,39]. Here, choline in the form of PC is an essential co-substrate of sphingomyelin synthase (EC 2.7.8.27) for ceramide clearance [40]. Given that decreased plasma choline and PC in CF patients reflect their deficiency in peripheral organs, choline deficiency may contribute to increased pulmonary ceramide levels.
Plasma choline and PC concentration were lower in adult CF patients than in healthy individuals, which is consistent with impaired choline status, due to increased fecal losses of biliary PC [8,13,27,41]. The correlation between body mass index (BMI) and lung function (FEV 1 ) suggests that nutrients and gastrointestinal function are important to the clinical course of these patients [2,3]. In this context C20:4 and C22:6 status and supplementation have been investigated extensively in CF. Whereas C22:6 supplementation in neutral lipid form did not convincingly improve clinical outcome [29,30], there is so far no focus on choline deficiency, impaired PC homeostasis, and their link to C20:4 and C22:6 metabolism, in relation to clinical parameters. The data of this study, however, indicate that in CF decreased plasma choline and total PC, and their link to LC-PUFA via C20:4-PC and C22:6-PC metabolism, specifically relates to lung function impairment and inflammation.
In contrast to decreased C18:2-containing PC, we did not find a significant alteration in the mean molar fractions of C20:4-PC or C22:6-PC, or in the ratio between C20:4-PC and C22:6-PC. Generally, C20:4 and C22:6 alterations in CF are highly variable, depending on age and model, and are explained by altered fat supply and absorption. Hepatic triglyceride accumulation with an altered fatty acid composition is believed to contribute as well [8,17,28,42]. However, C20:4 and C22:6 supply is not impaired in CF patients [28,42]. Triglycerides showed no differences in fatty acid composition and concentrations between CF patients and controls, with palmitic acid (C16:0) and C18:1 as major, and C20:4 and C22:6 as minor components (data not shown). Hence, the absolute decrease and high variability of LC-PUFA-PC in this study appears to relate to choline and PC metabolism rather than C20:4 and C22:6 availability. Consequently, the correlation between C20:4-PC and C22:6-PC values and clinical parameters suggest that impaired choline and PC homeostasis rather than LC-PUFA deficiency is relevant. In this context, it is noteworthy that correlations to FEV1 and IL-6 occur in a parallel fashion for both C20:4-PC and C22:6-PC. This is in clear contradiction to the hypothesis of increased C20:4 at the expense of C22:6 as the cause for clinical impairment [28]. In contrast, our data support the superior relevance of choline deficiency, impairing total and LC-PUFA-PC metabolism and clinical parameters.
The superior relevance of choline and PC homeostasis to extra-hepatic organ function is supported by data showing that in deficient mice choline and PC are redistributed from peripheral organs to the liver [43]. Our results are in line with this: the direct correlation of choline, total PC, C20:4-PC and C22:6-PC with lung function -and inversely with inflammation -suggests a general role of choline and PC status for the patients' clinical course. Importantly, patients were separable into two groups: four patients had higher choline, PC, C20:4-PC  Fig. 3B), the PC sub-group synthesized by the PEMT pathway, as demonstrated by [D 9 -methyl]choline labeling (Fig. 6D+F). As this pathway is the only source of (limited) endogenous choline synthesis [11], severity of choline deficiency in CF patients may depend on PEMT activity, which is highly variable among individuals [44,45]. This consequently means that the necessity and magnitude of any putative choline substitution of CF patients is variable. Moreover, it means that plasma choline and PC may be useful screening parameters related to clinical outcome in CF.

PC metabolism in CF plasma
To address characteristic differences of choline and PC metabolism and plasma kinetics we employed, for the first time in CF patients, in vivo labeling with [D 9 -methyl]choline [21,26]. This enabled us to investigate de novo synthesis of PC via direct [D 9 -methyl]choline incorporation, the major source of mono-and di-unsaturated PC in plasma, resulting in the formation of D 9 -labeled PC [21,26]. Additionally, [D 9 -methyl]choline labeling addresses PC synthesis from PE methylation by PEMT, which comprises about 30% of plasma PC and is the primary source of poly-unsaturated C20:4-PC and C22:6-PC: a fraction of administered [D 9 methyl]choline is oxidized to [D 9 -methyl]betaine, which then transfers a D 3 -methyl group to homocysteine via betaine homocysteine methyltransferase (EC: 2.1.1.5) to synthesize D 3methionine. This is subsequently activated to D 3 -S-adenosylmethionine (D 3 -SAM). D 3 -SAM is then used for sequential PE-methylation by PEMT, resulting in D 3 /D 6 -rather than D 9 -labeled PC [21,46,47].
After [D 9 -methyl]choline chloride infusion, [D 9 -methyl]choline enrichment of PC was identical in CF and control subjects. It is not possible to determine deuterium enrichment of intracellular precursors of PC synthesis for ethical reasons. As D 9 -enrichment in plasma choline was identical in CF and controls, and given that this is representative for the intracellular space, data indicate unchanged de novo PC synthesis in CF. However, this only means that synthesis is identical relative to the respective hepatic PC pools in the enrolled individuals. D 3/6 -enrichment of PC representing the PEMT pathway, however, was lower in CF compared to controls, although the estimated D 3 -enrichment of SAM was identical in both groups. In controls, it was about 30% of total deuterium-enrichment of plasma PC, which equals the estimated contribution of this pathway to normal hepatic PC metabolism [48], whereas it was less in CF patients. Hence, our data show that the PEMT pathway, was decreased in the enrolled 3 CF patients compared to controls. Inter-individual differences of PEMT activity, may explain for the differences in choline and PC homeostasis and clinical parameters across CF patients (see above).
Total concentrations of both newly synthesized D 9 -and D 3/6 -PC were lower in CF plasma. Substrate (choline, SAM) deficiency, the priority of biliary PC secretion over that of VLDL-PC [11], or their combination may contribute here. These data are consistent with the general impairments in choline and PC homeostasis we found in the whole study group, and may therefore be representative for CF patients. Importantly, choline substitution improves the methyl group status in CF patients [49]. However, effects on PC metabolism and PEMT activity were not investigated in that study. The hepatic status of the patients participating in [D 9 -methyl]choline labeling was highly different, whereas metabolic alterations were similar. We therefore hypothesize that decreased hepatic PC secretion into plasma may be a general phenomenon in CF patients due to impaired choline status, which also explains for decreased plasma cholesterol [50]. Nevertheless, further mechanisms, like accelerated PC turnover in plasma due to peripheral consumption or hepatic uptake of high-density lipoproteins (HDL), being rich in cholesterol as well as in PC [19], may contribute. In essence, it will be important to assess in larger studies whether strategies to supplement choline and replenish the pool of one-carbon-units will normalize plasma choline and PC homeostasis, and will improve the clinical status in CF. Sub-grouping of PC [27] showed that only D 9 -C18:1-PC kinetics was unchanged. This is consistent with increased PC16:0/18:1 fractions -methyl]choline labeling were of minor extent. The complex interactions of differential plasma turnover, trafficking of individual PC species to bile or VLDL, HDL uptake and the usage of LC-PUFA-PE components for the PEMT pathway [11,21,51] lead to a complex situation, where stable isotope labeling and MS/MS analysis proved more sensitive than HPLC. However, all these aspects will have to be investigated in a larger number of CF patients in future.

Conclusion and Clinical Perspective
Our data point to a pathophysiological concept of impaired hepatic PC synthesis and secretion, based on choline deficiency. This contributes to the clinical status of CF patients, as plasma choline and PC concentrations directly correlate with lung function and inversely with inflammation. Hence, plasma choline and PC analysis may be clinically useful screening parameters. Inter-individual differences in PEMT activity, responsible for C22:6 and C20:4-PC and (limited) endogenous choline synthesis, may contribute to the overall choline status of these patients. Research is needed to specify whether clinical status can be improved by choline substitution, which is momentarily addressed in a pilot study.