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.

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

Deuterated choline-d(9) 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.

A validated bioanalytical method in mouse, rat, rabbit and human plasma for the quantification of one of the steroid glycosides found in Hoodia gordonii extract.

Hoodia gordonii extract contains steroid glycosides, fatty acids, plant sterols and polar organic material. Certain steroid glycosides show appetite suppressant activities following oral ingestion. This study describes the validation of a bioanalytical method for the quantification of one of the steroid glycosides, H.g.-12 (≈ 10% (w/w) of the extract), in mouse, rat, rabbit and human plasma. The method utilises a liquid-liquid extraction with methyl-tert-butyl ether followed by chromatographic separation on a 2.1 × 50 mm C(18) Genesis high performance liquid chromatography (HPLC) column and detection on a triple quadrupole mass spectrometer. Detection of H.g.-12 and its stable isotope internal standards is performed using positive TurboIonspray™ ionisation in multiple reaction monitoring mode. The validation procedure demonstrated assay sensitivity, linearity, accuracy, precision and selectivity over the calibration range of 0.5-150 ng/mL in human plasma (500 µL sample volume), 1.0-100 ng/mL in rat and rabbit plasma (150 µL sample volume) and 1.0-250 ng/mL in mouse plasma (150 µL sample volume) with good recoveries (≥ 77%). H.g.-12 was stable in plasma for ≥ 6 months at -20°C, for up to 4h at ambient temperature (ca22°C) and after 3 freeze-thaw cycles. Plasma extracts were stable for up to 24h at ambient temperature.

Utilization of DBS within drug discovery: a simple 2D-LC-MS/MS system to minimize blood- and paper-based matrix effects from FTA elute™ DBS.

BACKGROUND: Dried blood spot-based bioanalysis potentially introduces novel matrix effects that need to be eliminated or controlled. Within nonregulatory drug discovery these can be defined as ≤20% and ≤30% for nominal peak area, respectively. RESULTS: Controlling matrix effects for a panel of compounds by simple 1D-HPLC-MS/MS was not achievable and the optimization of 2D-HPLC-MS/MS is reported here. Simple inclusion of a 'trapping' stage was not sufficient to improve matrix effects and optimization of the reconstitution solvent, reconstitution volume and injection volume was required for a generic system to be developed. CONCLUSION: A generic 2D-LC-MS/MS system has been developed that eliminates paper-based matrix effects and eliminates or controls dried blood spot matrix effects for a panel of compounds extracted from FTA Elute™ with methanol.

Bioanalytical determination of unstable endogenous small peptides: RFRP3 and its metabolites in rat blood.

BACKGROUND: Targeting the gonadotropin-releasing hormone pathway for the treatment of endometriosis leads to an interest in monitoring for endogenous modulators of this pathway (RFRP3 and kisspeptin) as baseline controls for treatment development. RESULTS: Stabilization of RFRP3 was shown to be extremely difficult in a highly enzymatically active matrix, such as rat blood. Sample denaturing with solvent at collection was necessary due to enzyme inhibition being unsuccessful at stabilization leading to difficulties in sample processing. Monitoring multiple fragments formed in blood can aid in profiling these peptides once in-source conversion is controlled. CONCLUSION: generic high-sensitivity LC-MS/MS assay was developed for RFRP3 and the fragments formed from it in whole blood. Use of 2D chromatography circumvents concentration and retention issues related to small fragments with a normal flow setup, making a more open-access approach feasible.

Utilization of DBS within drug discovery: development of a serial microsampling pharmacokinetic study in mice.

BACKGROUND: Dried blood spots (DBS) are rapidly gaining a foothold in the pharmaceutical industry. However, applications in exploratory drug discovery are limited mainly owing to method development time. The development of a generic DBS assay is presented with its application in serial microsampling from mice. RESULTS: A generic 'fit-for-purpose' assay was developed on FTA(®) Elute, which allowed 90% of compounds tested to reach sensitivity levels of 1 ng/ml. A ten-time point serial mouse pharmacokinetic study was conducted using 20-µl microsamples and DBS. Application of generic 'fit-for-purpose' approach did not compromise data delivery or quality. CONCLUSIONS: Serial microsampling and DBS in exploratory mouse pharmacokinetic has been shown to provide superior data quality when compared with traditional plasma-based composite studies.

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 (22:6(n-3)) and increased arachidonate (20:4(n-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 cftr(tm1HGU/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 22:6(n-3) in any phospholipid class from lung, pancreas, or liver from mice with the cftr(tm1HGU/tm1HGU). Instead, the concentration of 20:4(n-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 cftr(tm1HGU/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 22:6(n-3)-containing oils to children with CF can be fully evaluated.