Wolfgang Bernhard

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

Phosphatidylcholine (PC) synthesis by the direct cytidine diphosphate choline (CDP-choline) pathway in rat liver generates predominantly mono- and di-unsaturated molecular species, while polyunsaturated PC species are synthesized largely by the phosphatidylethanolamine-N-methyltransferase (PEMT) pathway. Although altered PC synthesis has been suggested to contribute to development of hepatocarcinoma and nonalcoholic steatohepatitis, analysis of the specificity of hepatic PC metabolism in human patients has been limited by the lack of sensitive and safe methodologies. Here we incorporated a deuterated methyl-d9-labled choline chloride, to quantify biosynthesis fluxes through both of the PC synthetic pathways in vivo in human volunteers and compared these fluxes with those in mice. Rates and molecular specificities of label incorporated into mouse liver and plasma PC were very similar and strongly suggest that label incorporation into human plasma PC can provide a direct measure of hepatic PC synthesis in human subjects. Importantly, we demonstrate for the first time that the PEMT pathway in human liver is selective for polyunsaturated PC species, especially those containing docosahexaenoic acid. Finally, we present a multiple isotopomer distribution analysis approach, based on transfer of deuterated methyl groups to S-adenosylmethionine and subsequent sequential methylations of PE, to quantify absolute flux rates through the PEMT pathway that are applicable to studies of liver dysfunction in clinical studies.

COMPOSITION OF PHOSPHOLIPID CLASSES AND PHOSPHATIDYLCHOLINE MOLECULAR SPECIES OF GASTRIC MUCOSA AND MUCUS

Phospholipids have been proposed to protect the gastric mucosa by forming a proton-repellant hydrophobic layer on the gastric luminal surface, acting as a so-called gastric surfactant. The composition of this hydrophobic phospholipid layer has not previously been analysed in detail. Therefore, we measured the composition of phospholipid classes and phosphatidylcholine (PC) molecular species in gastric mucosa and mucus of rats and pigs using high resolution HPLC techniques. The predominant phospholipids of both mucosa and mucus were PC and phosphatidylethanolamine (PE). Little phosphatidylglycerol was present. The most abundant PC species of rat mucosa were PC16:0/18:1, PC16:0/18:2, PC16:0/20:4 and PC18:0/20:4. Pig mucosa also contained PC16:0/18:1, PC16:0/18:2, and PC18:0/20:4, but was poor in PC16:0/20:4. Dipalmitoyl-PC (PC16:0/16:0), the surface-active component of pulmonary surfactant, comprised only 6.42 +/- 0.33% of total PC in rat mucosa and only 5.50 +/- 1.46% of total PC in pig mucosa. Gastric mucus, isolated from both rat and pig, contained largely PC16:0/18:1 and PC16:0/18:2. The content of PC16:0/16:0 was even lower in mucus than in mucosal PC (rat 2.86 +/- 0.40%, P < 0.01; pig 1.92 +/- 0.55%, P < 0.05). We conclude that, in contrast to pulmonary surfactant, any surfactant function of the hydrophobic barrier of the stomach is unlikely to be mediated by PC16:0/16:0.

Lipidomics of cellular and secreted phospholipids from differentiated human fetal type II alveolar epithelial cells

Maturation of fetal alveolar type II epithelial cells in utero is characterized by specific changes to lung surfactant phospholipids. Here, we quantified the effects of hormonal differentiation in vitro on the molecular specificity of cellular and secreted phospholipids from human fetal type II epithelial cells using electrospray ionization mass spectrometry. Differentiation, assessed by morphology and changes in gene expression, was accompanied by restricted and specific modifications to cell phospholipids, principally enrichments of shorter chain species of phosphatidylcholine (PC) and phosphatidylinositol, that were not observed in fetal lung fibroblasts. Treatment of differentiated epithelial cells with secretagogues stimulated the secretion of functional surfactant-containing surfactant proteins B and C (SP-B and SP-C). Secreted material was further enriched in this same set of phospholipid species but was characterized by increased contents of short-chain monounsaturated and disaturated species other than dipalmitoyl PC (PC16:0/16:0), principally palmitoylmyristoyl PC (PC16:0/14:0) and palmitoylpalmitoleoyl PC (PC16:0/16:1). Mixtures of these PC molecular species, phosphatidylglycerol, and SP-B and SP-C were functionally active and rapidly generated low surface tension on compression in a pulsating bubble surfactometer. These results suggest that hormonally differentiated human fetal type II cells do not select the molecular composition of surfactant phospholipid on the basis of saturation but, more likely, on the basis of acyl chain length.

Molecular Species Compositions of Lung and Pancreas Phospholipids in the cftr tm1HGU/tm1HGU Cystic Fibrosis Mouse

Fatty acid analysis of phospholipid compositions of lung and pancreas cells from a cystic fibrosis transmembrane regulator (CFTR) negative mouse (cftr−/−)suggested that a decreased concentration of docosahexaenoate (22:6n-3) and increased arachidonate (20:4n-6) may be related to the disease process in cystic fibrosis (CF). Consequently, we have determined compositions of the major phospholipids of lung, pancreas, liver, and plasma from a different mouse model of CF, the cftrtm1HGU/tm1HGU mouse, compared with ZTM:MF-1 control mice. Electrospray ionization mass spectrometry permitted the quantification of all of the individual molecular species of phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylglycerol (PtdGly), phosphatidylserine (PtdSer), and phosphatidylinositol (PtdIns). There was no deficiency of 22:6n-3 in any phospholipid class from lung, pancreas, or liver from mice with the cftrtm1HGU/tm1HGU. Instead, the concentration of 20:4n-6 was significantly decreased in plasma PtdCho species and in pancreas and lung species of PtdEtn, PtdSer, and PtdIns. These results demonstrate the variability of membrane phospholipid compositions in different mouse models of CF and suggest that in cftrtm1HGU/tm1HGU mice, the apparent deficiency was of 20:4n-6- rather than of 22:6n-3–containing phospholipid species. They highlight a need for detailed phospholipid molecular species analysis of cells expressing mutant CFTR from children with CF before the therapeutic effects of administering high doses of 22:6n-3–containing oils to children with CF can be fully evaluated.

Differential effect of surfactant and its saturated phosphatidylcholines on human blood macrophages

Blood monocyte-derived macrophages invading the alveolus encounter pulmonary surfactant, a phospholipoprotein complex that changes composition during lung development. We tested the hypothesis that characteristic phosphatidylcholine (PC) components differentially influence macrophage phenotype and function, as determined by phagocytosis of green fluorescent protein-labeled Escherichia coli and αCD3-induced T cell proliferation. Human macrophages were exposed to surfactant (Curosurf®), to two of its characteristic phosphadidylcholine (PC) components (dipalmitoyl-PC and palmitoylmyristoyl-PC), and to a ubiquituous PC (palmitoyloleoyl-PC) as control. Interaction of Curosurf and PC species with macrophages was assessed using Lissamine™-dihexadecanoyl-phosphoethanolamine-labeled liposomes. Curosurf and both saturated surfactant PC species downregulated CD14 expression and upregulated CD206. HLA-DR and CD80 were upregulated by Curosurf and palmitoylmyristoyl-PC, whereas dipalmitoyl-PC showed no effect. The latter upregulated TLR2 and TLR4 expression, whereas Curosurf and palmitoylmyristoyl-PC had no effect. PC species tested were incorporated in comparable amounts by macrophages. Curosurf and PC species inhibited phagocytosis of E. coli. Scavenger receptor CD36, CD68, SR-A, and LOX-1 mRNA expression was upregulated by Curosurf, whereas PC species only upregulated SR-A. Curosurf and palmitoylmyristoyl-PC inhibited αCD3-induced T cell proliferation by 50%, whereas dipalmitoyl-PC and palmitoyloleoyl-PC showed no effect. These data identify individual surfactant PC species as modifiers of macrophage differentiation and suggest differential effects on innate and adaptive immune functions.

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 organisms 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.

A Historic Cohort Study on Accelerated Advancement of Enteral Feeding Volumes in Very Premature Infants

Background: The optimal rate of enteral feeding (EF) advancement in very low birth weight infants is under debate. Objectives: To evaluate the effects of accelerated EF advancement on the time to full enteral feeds, on early postnatal growth as well as on the frequency of necrotizing enterocolitis (NEC) and focal intestinal perforation (FIP) in very premature infants. Methods: In a retrospective single-center historic cohort study, infants with a gestational age <32 weeks at birth and birth weight <1,500 g, born between January 1, 2006, and December 31, 2007 (n = 136), were compared with infants born between January 1, 2010, and December 31, 2010 (n = 88). In 2006/2007, enteral feeds were initiated on day 1 with 10–15 ml/kg/day and advanced by 15–20 ml/kg/day. In 2010, enteral feeds were initiated with 20 ml/kg/day on day 1 and advanced by 25–30 ml/kg/day. Full enteral feeds were defined as ≥140 ml/kg/day. Data are presented as median (P25–P75). Results: The time to establish full enteral feeds was shorter in 2010: 8 (7–11) days in 2006/2007 versus 6 (5–9) days in 2010. The incidences of NEC and FIP were 2.7 and 4.1% in 2006/2007 and 3.3 and 2.2% in 2010, respectively. Weight gain was not affected by the rate of EF advancement. Higher parenteral protein intake during week 1 in 2006/2007 was associated with better head circumference growth. Conclusions: The new approach was associated with a significantly shorter period to establish full enteral feeds. No difference in the incidence of FIP or NEC was observed; however, the study was underpowered to detect small but possibly important differences.

Myristate is selectively incorporated into surfactant and decreases dipalmitoylphosphatidylcholine without functional impairment

Lung surfactant mainly comprises phosphatidylcholines (PC), together with phosphatidylglycerols and surfactant proteins SP-A to SP-D. Dipalmitoyl-PC (PC16:0/16:0), palmitoylmyristoyl-PC (PC16:0/14:0), and palmitoylpalmitoleoyl-PC (PC16:0/16:1) together comprise 75-80% of surfactant PC. During alveolarization, which occurs postnatally in the rat, PC16:0/14:0 reversibly increases at the expense of PC16:0/16:0. As lipoproteins modify surfactant metabolism, we postulated an extrapulmonary origin of PC16:0/14:0 enrichment in surfactant. We, therefore, fed rats (d19-26) with trilaurin (C12:0(3)), trimyristin (C14:0(3)), tripalmitin (C16:0(3)), triolein (C18:1(3)) or trilinolein (C18:2(3)) vs. carbohydrate diet to assess their effects on surfactant PC composition and surface tension function using a captive bubble surfactometer. Metabolism was assessed with deuterated C12:0 (ω-d(3)-C12:0) and ω-d(3)-C14:0. C14:0(3) increased PC16:0/14:0 in surfactant from 12 ± 1 to 45 ± 3% and decreased PC16:0/16:0 from 47 ± 1 to 29 ± 2%, with no impairment of surface tension function. Combined phospholipase A(2) assay and mass spectrometry revealed that 50% of the PC16:0/14:0 peak comprised its isomer 1-myristoyl-2-palmitoyl-PC (PC14:0/16:0). While C12:0(3) was excluded from incorporation into PC, it increased PC16:0/14:0 as well. C16:0(3), C18:1(3), and C18:2(3) had no significant effect on PC16:0/16:0 or PC16:0/14:0. d(3)-C14:0 was enriched in lung PC, either via direct supply or via d(3)-C12:0 elongation. Enrichment of d(3)-C14:0 in surfactant PC contrasted its rapid turnover in plasma and liver PC, where its elongation product d(3)-C16:0 surmounted d(3)-C14:0. In summary, high surfactant PC16:0/14:0 during lung development correlates with C14:0 and C12:0 supply via specific C14:0 enrichment into lung PC. Surfactant that is high in PC16:0/14:0 but low in PC16:0/16:0 is compatible with normal respiration and surfactant function in vitro.

rhKGF stimulates lung surfactant production in neonatal rats in vivo

Surfactant deficiency and bronchopulmonary dysplasia (BPD), major obstacles in preterm infants, are addressed with pre‐ and postnatal glucocorticoids which also evoke harmful catabolic side‐effects. Keratinocyte growth factor (KGF) accelerates surfactant production in fetal type II pneumocytes (PN‐II), protects epithelia from injury and is deficient in lungs developing BPD, highlighting its potential efficacy in neonates. Neonatal rats were treated with recombinant human (rh)KGF, betamethasone, or their combination for 48 hr prior to sacrifice after which body weight, surfactant, and tissue phosphatidylcholines (PC) were investigated at postnatal d3, d7, d15, and d21. Pneumocyte proliferation, surfactant protein (SP) expression and SP‐B/C in lung lavage fluid (LLF) were also determined at d7 and d21 to identify broader surfactant changes occurring at the beginning and end of the initial alveolarization phase. While all treatments increased secreted surfactant PC, BM compromised animal growth whereas rhKGF did not. At d3 rhKGF was more effective in male compared to female rats. Single treatments became less effective towards d21. Neither treatment altered PC composition in LLF. BM inhibited PN‐II proliferation and increased surfactant PCs at the expense of tissue PCs. rhKGF however increased surfactant PCs without decreasing other PC species. Whereas SP‐B/C gene expression was induced by all treatments, the changes in secreted SP‐B/C mirrored those observed for surfactant PC. Our results encourage investigation of the mechanisms by which rhKGF improves surfactant homoeostasis, and detailed examination of its efficacy in neonatal lung injury models with a view to implementing it as a non‐catabolic surfactant‐increasing therapeutic in neonatal intensive care. Pediatr. Pulmonol. 2011; 46:883–895.

Active surfactant in pharyngeal aspirates of term neonates: lipid biochemistry and surface tension function

Alveolar surfactant is well known for its ability to reduce minimal surface tension at the alveolar air–liquid interface to values below 5 mN m−1. In addition, it has been suggested that surfactant is also present in the airways, particularly in the perinatal period. We isolated surfactant from pharyngeal aspirates obtained from 33 neonates immediately after delivery and analysed it for both phospholipid (PL) composition and surface tension function. PL classes and phosphatidylcholine (PC) molecular species were determined by normal and reversed‐phase high‐performance liquid chromatography (HPLC), respectively. Static and dynamic surface properties of the surfactant were studied in a pulsating bubble surfactometer. Sample volume was 1.3 ± 0.5 mL (mean ± SD) with a total amount of 2.5 ± 1.3 μmol of PL and a concentration of 2.1 ± 1.0 μmol mL−1 PL. HPLC analyses of PL classes revealed a composition identical with surfactant prepared from alveolar washes, i.e. PC 83.6 ± 2.1%, sphingomyelin 1.4 ± 0.5%, phosphatidylglycerol 8.1 ± 1.6%, phosphatidylethanolamine 2.1 ± 0.5% and phosphatidylinositol 2.6 ± 1.1%. Thin‐layer chromatography showed almost identical results but was more time‐consuming and needed more material for analysis. Analysis of PC molecular species revealed a composition typical of human alveolar surfactant with 54.7 ± 3.9% dipalmitoyl PC, 10.3 ± 1.9% palmitoyloleoyl PC and 9.1 ± 1.5% palmitoylmyristoyl PC. Minimal surface tension fell to values below 5 mN m−1 within 5 min of cycling in all subjects. The methods used in this study allowed for complete PL and surface tension analyses of surfactant obtained during routine pharyngeal suctioning after delivery at term. Whether they are also applicable to preterm neonates with respiratory distress remains to be determined.