RESUMO
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-D(9)-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.
Assuntos
Citidina Difosfato Colina/metabolismo , Fosfatidilcolinas/biossíntese , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Adulto , Animais , Colina/metabolismo , Deutério , Feminino , Humanos , Fígado/metabolismo , Pulmão/metabolismo , Masculino , Camundongos , Fosfatidiletanolamina N-Metiltransferase/sangueRESUMO
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.
Assuntos
Lesão Pulmonar Aguda/fisiopatologia , Inflamação/diagnóstico , Fosfolipídeos/análise , Surfactantes Pulmonares/análise , Biomarcadores/metabolismo , Líquido da Lavagem Broncoalveolar/química , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Inflamação/fisiopatologia , Unidades de Terapia Intensiva Pediátrica , Interleucina-8/metabolismo , Intubação Intratraqueal , Lipoproteínas/metabolismo , Masculino , Estudos Prospectivos , Síndrome do Desconforto Respiratório/fisiopatologiaRESUMO
INTRODUCTION: Defining research priorities in intensive care is key to determining appropriate allocation of funding. Several topics were identified from the 2014 James Lind Alliance priority setting exercise conducted with the Intensive Care Society. The James Lind Alliance process included significant (and vital) patient/public contribution, but excluded professionals without a bedside role. As a result it may have failed to identify potential early-stage translational research topics, which are more likely identified by medical and/or academic members of relevant specialist basic science groups. The objective of the present project was to complement the James Lind Alliance project by generating an updated list of research priorities by facilitating academic research input. METHOD: A survey was conducted by the National Institute for Health Research (NIHR) to identify the key research priorities from intensive care clinicians, including allied health professionals and academics, along with any evolving themes arising from translational research. Feasibility of all identified topics were then discussed and allocated to themes by a joint clinical academics/NIHR focus group. RESULTS: The survey was completed by 94 intensive care clinicians (including subspecialists), academics and allied health professions. In total, 203 research questions were identified, with the top five themes focusing on: appropriate case selection (e.g. who and when to treat; 24%), ventilation (7%), sepsis (6%), delirium (5%) and rehabilitation (5%). DISCUSSION: Utilising a methodology distinct from that employed by the James Lind Alliance process, from a broad spectrum of intensive care clinicians/scientists, enabled identification of a variety of priority research areas. These topics can now inform not only the investigator-led research agenda, but will also be considered in due course by the NIHR for potential future funding calls.
RESUMO
Stable isotope labelling of lipid precursors coupled with mass spectrometry-based lipidomic analyses and determination of isotope enrichment in substrate, intermediate and product pools provide the parameters needed to determine absolute flux rates through lipid pathways in vivo. Here, as an illustration of the power of such analyses we investigated lung phosphatidylcholine (PC) synthesis in Surfactant Protein-D (SP-D) null mice. These animals develop emphysema, foamy alveolar macrophages and an alveolar lipoproteinosis with increasing age. We used the incorporation of methyl-9-[(2)H] choline chloride coupled with ESI-MS/MS to quantify absolute rates of lung surfactant PC synthesis and secretion in an SP-D(-/-) mouse model, together with an analysis of the molecular specificity of lung PC synthesis. PC synthetic rates were comparable in control (0.52 µmol/lung/h) and SP-D(-/-) (0.69 µmol/lung/h) mice, as were rates of surfactant PC secretion (29.8 and 30.6 nmol/lung/h, respectively). Increased lung PC in the SP-D(-/-) mouse was due to impaired catabolism, with a rate of accumulation of 0.057 µmol/lung/h. The relatively low rates of surfactant PC secretion compared with total lung PC synthesis were compatible with a suggested ABCA1-mediated basolateral lipid efflux from alveolar type II epithelial cells. Finally, PC molecular species analysis suggested that a proportion of newly synthesised PC is secreted rapidly into the lung air spaces in both control and SP-D(-/-) mice before significant PC acyl remodelling occurs.