Graeme T. Clark

Highly saturated endonuclear phosphatidylcholine is synthesized in situ and colocated with CDP-choline pathway enzymes.

Chromatin-associated phospholipids are well recognized. A report that catalytically active endonuclear CTP:choline-phosphate cytidylyltransferase α is necessary for cell survival questions whether endonuclear, CDP-choline pathway phosphatidylcholine synthesis may occur in situ. We report that chromatin from human IMR-32 neuroblastoma cells possesses such a biosynthetic pathway. First, membrane-free nuclei retain all three CDP-choline pathway enzymes in proportions comparable with the content of chromatin-associated phosphatidylcholine. Second, following supplementation of cells with deuterated choline and using electrospray ionization mass spectrometry, both the time course and molecular species labeling pattern of newly synthesized endonuclear and whole cell phosphatidylcholine revealed the operation of spatially separate, compositionally distinct biosynthetic routes. Specifically, endogenous and newly synthesized endonuclear phosphatidylcholine species are both characterized by a high degree of diacyl/alkylacyl chain saturation. This unusual species content and synthetic pattern (evident within 10 min of supplementation) are maintained through cell growth arrest by serum depletion and when proliferation is restored, suggesting that endonuclear disaturated phosphatidylcholine enrichment is essential and closely regulated. We propose that endonuclear phosphatidylcholine synthesis may regulate periodic nuclear accumulations of phosphatidylcholine-derived lipid second messengers. Furthermore, our estimates of saturated phosphatidylcholine nuclear volume occupancy of around 10% may imply a significant additional role in regulating chromatin structure.

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.

A comparison of the molecular specificities of whole cell and endonuclear phosphatidylcholine synthesis.

Deuterated choline‐d9 labelling of IMR‐32 cells enabled comparison of the molecular specificities of whole cell and endonuclear phosphatidylcholine synthesis after 96 h polyunsaturated fatty acid supplementation. Surprisingly, while cell phosphatidylcholine synthesis and remodelling reflected a pattern of polyunsaturated fatty acid accretion, the saturated endonuclear phosphatidylcholine pool was only transiently labelled with polyunsaturates. Periodic endonuclear accumulations of the lipid second messenger diacylglycerol, mobilised from unsaturated phosphatidylinositol or saturated phosphatidylcholine, accompany cell proliferation. Non‐specific incorporation into endonuclear phosphatidylcholine and selective removal or remodelling of unsaturated molecular species may form part of a single ‘off switch’ recycling all endonuclear diacylglycerol accumulations.

Surfactant phospholipids, surfactant proteins, and inflammatory markers during acute lung injury in children.

Objective: To explore the pathophysiology of acute lung injury in children. Design: Prospective cohort study. Setting: Regional University Hospital, pediatric intensive care unit. Patients: Children without a preexisting lung injury who developed acute lung injury and were intubated were eligible for the study. Children without lung injury and intubated for minor surgical procedures acted as controls. Interventions: Bronchoalveolar lavage fluid and blood were collected on days 1 to 4, weekly, and immediately before extubation during acute lung injury. Molecular species compositions of phosphatidylcholine were determined by electrospray ionization mass spectrometry of lipid extracts of bronchoalveolar lavage fluid supernatants. Surfactant proteins A, B, and D and interleukin-8 were measured in bronchoalveolar lavage fluid and plasma by enzyme-linked immunosorbent assay and Western blotting. Measurements and Main Results: Eighteen children with acute lung injury were enrolled in the study and compared with eight controls. In children with acute lung injury, there were significant changes in the bronchoalveolar lavage fluid phosphatidylcholine species. Bronchoalveolar lavage fluid dipalmitoyl phosphatidylcholine (PC 16:0/16:0) and palmitoyl-myristoyl phosphatidylcholine (PC 16:0/14:0) significantly deceased during acute lung injury (p < .001 and p < .001, respectively), whereas oleoyl-linoleoyl PC (18:1/18:2), palmitoyl-linoleoyl PC (16:0/18:2) and stearoyl-linoleoyl PC (18:0/18:2) characteristic of plasma PC were significantly increased (p < .05, p < .02, and p < .05 respectively), as well as palmitoyl-oleoyl PC (16:0/18:1), and stearoyl-arachidonoyl PC (18:0/20:4) which are characteristic of cell membranes (p < .02, and p < .02, respectively). There were no significant changes to bronchoalveolar lavage fluid, surfactant protein A or B levels compared with controls during acute lung injury, whereas bronchoalveolar lavage fluid, surfactant protein D, and interleukin-8 levels significantly increased (p < .05 and p < .02, respectively). In plasma during acute lung injury, there were significant increases in surfactant proteins A, B, and D, and interleukin-8 (p < .001, p < .001, p < .05, and p < .001, respectively). Conclusion: Changes to the phosphatidylcholine profile, surfactant proteins, and inflammatory markers of bronchoalveolar lavage fluid and plasma in children with acute lung injury are consistent with an alveolar/blood leakage and inflammatory cell membrane degradation products. These changes are due to alveolar capillary membrane damage and cellular infiltration.

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.

Lipidomic analysis of the molecular specificity of a cholinephosphotransferase in situ.

Dynamic lipidomics using ESI-MS (tandem electrospray ionization mass spectrometry) of 9-deuterated choline (choline-d(9)) incorporation into mammalian cell PtdCho (phosphatidylcholine) permits assessment of the molecular specificity of synthesis. Bulk cell PtdCho synthesis occurs in spatially distinct locations, using separate CPTs (1,2 diacylglycerol CDP:choline cholinephosphotransferases). We assessed whether in vitro molecular selectivity of DAG (diacylglycerol) incorporation between CPTs is manifest in situ, by monitoring choline-d(9) incorporation into PtdCho and lyso-PtdCho molecular species over 3 h in control cells and in CHO-K1 cells overexpressing hCEPT1. Compared with controls, the basal molecular species composition of hCEPT1 overexpressors was significantly enriched in arachidonate. This was not due to net accretion of cellular PtdCho arguing against effects of inadequate unsaturated PtdCho degradation or remodelling. Rather, time-course analyses of PtdCho and lyso-PtdCho pools showed that both arachidonate-containing DAG incorporation and turnover of PtdCho is increased in hCEPT1 overexpressors. Increased choline-d(9) incorporation into arachidonyl lyso-PtdCho shows that both phospholipase A(1)- and A(2)-mediated turnover is involved. Spatially distinct molecular specificity of DAG incorporation into cellular PtdCho at the level of hCEPT1 exists in situ.

Electrospray ionisation mass spectrometry analysis of differential turnover of phosphatidylcholine by human blood leukocytes

Synthesis and turnover of membrane phospholipids is essential for cell growth and function, and hydrolysis of membrane phospholipid is central to many intracellular signalling mechanisms. Hydrolysis of phosphatidylcholine (PC) is a major signalling mechanism of neutrophils, leukocytes that phagocytose and kill bacteria as part of the innate immune response, generating phosphatidic acid, diacylglycerol and arachidonate-derived lipid second messengers. We describe here the application of tandem MS/MS electrospray ionisation mass spectrometry to the analysis of molecular patterns of PC synthesis by blood neutrophil and lymphocyte cells from healthy volunteers. This technique combined incorporation of the headgroup choline, methyl-labelled with deuterium (methyl-d9-choline), with precursor scans of diagnostic labelled and native fragment ions. The technique was very sensitive, permitting detection of d9 enrichment <0.01%. Results showed that the two different cell types maintained distinct molecular species compositions of PC, even though they were exposed to the same nutrient supply in blood. Moreover, while the pattern of lymphocyte PC synthesis directly mirrored composition, the fractional synthesis of arachidonoyl (C20∶4,n-6)-containing PC species in neutrophils was greatly enhanced compared with composition. This increased turnover of arachidonoyl species in neutrophils may be related to the active synthesis of eicosanoids and other arachidonoyl-derived mediators in this cell type.