Plasma lipidomics reveals potential prognostic signatures within a cohort of cystic fibrosis patients
2011; Elsevier BV; Volume: 52; Issue: 5 Linguagem: Inglês
10.1194/jlr.p013722
ISSN1539-7262
AutoresMario Ollero, Giuseppe Astarita, Ida Chiara Guerrera, Isabelle Sermet‐Gaudelus, Stéphanie Trudel, Daniele Piomelli, Aleksander Edelman,
Tópico(s)Advanced Chemical Sensor Technologies
ResumoCystic fibrosis (CF) is associated with abnormal lipid metabolism. We have recently shown variations in plasma levels of several phosphatidylcholine (PC) and lysophopshatidylcholine (LPC) species related to disease severity in CF patients. Here our goal was to search for blood plasma lipid signatures characteristic of CF patients bearing the same mutation (F508del) and different phenotypes, and to study their correlation with forced expiratory volume in 1 s (FEV1) and Pseudomonas aeruginosa chronic infection, evaluated at the time of testing (t = 0) and three years later (t = 3). Samples from 44 F508del homozygotes were subjected to a lipidomic approach based on LC-ESI-MS. Twelve free fatty acids were positively correlated with FEV1 at t = 0 (n = 29). Four of them (C20:3n-9, C20:5n-3, C22:5n-3, and C22:6n-3) were also positively correlated with FEV1 three years later, along with PC(32:2) and PC(36:4) (n = 31). Oleoylethanolamide (OEA) was negatively correlated with FEV1 progression (n = 17). Chronically infected patients at t = 0 showed lower PC(32:2), PC(38:5), and C18:3n-3 and higher cholesterol, cholesterol esters, and triacylglycerols (TAG). Chronically infected patients at t = 3 showed significantly lower levels of LPC(18:0). These results suggest a potential prognostic value for some lipid signatures in, to our knowledge, the first longitudinal study aimed at identifying lipid biomarkers for CF. Cystic fibrosis (CF) is associated with abnormal lipid metabolism. We have recently shown variations in plasma levels of several phosphatidylcholine (PC) and lysophopshatidylcholine (LPC) species related to disease severity in CF patients. Here our goal was to search for blood plasma lipid signatures characteristic of CF patients bearing the same mutation (F508del) and different phenotypes, and to study their correlation with forced expiratory volume in 1 s (FEV1) and Pseudomonas aeruginosa chronic infection, evaluated at the time of testing (t = 0) and three years later (t = 3). Samples from 44 F508del homozygotes were subjected to a lipidomic approach based on LC-ESI-MS. Twelve free fatty acids were positively correlated with FEV1 at t = 0 (n = 29). Four of them (C20:3n-9, C20:5n-3, C22:5n-3, and C22:6n-3) were also positively correlated with FEV1 three years later, along with PC(32:2) and PC(36:4) (n = 31). Oleoylethanolamide (OEA) was negatively correlated with FEV1 progression (n = 17). Chronically infected patients at t = 0 showed lower PC(32:2), PC(38:5), and C18:3n-3 and higher cholesterol, cholesterol esters, and triacylglycerols (TAG). Chronically infected patients at t = 3 showed significantly lower levels of LPC(18:0). These results suggest a potential prognostic value for some lipid signatures in, to our knowledge, the first longitudinal study aimed at identifying lipid biomarkers for CF. ERRATAJournal of Lipid ResearchVol. 53Issue 6PreviewAn affiliation for Dr. Daniele Piomelli, Italian Institute of Technology, was inadvertently omitted on the following papers Dr. Piomelli co-authored: Full-Text PDF Open Access Cystic fibrosis (CF) is a genetic disease attributed to mutations in the CFTR gene, coding for a widely expressed chloride channel (1Riordan J.R. Rommens J.M. Kerem B. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.L. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA.Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5977) Google Scholar). The disease phenotype is mostly circumscribed to the digestive and respiratory systems. The former mainly consists of intestinal obstruction and meconium ileus, pancreatic malfunction, and intestinal malabsorption. The latter leads to chronic obstructive pulmonary disease with recurrent infections and exacerbations, ultimately resulting in respiratory insufficiency and death (2Ollero M. Brouillard F. Edelman A. Cystic fibrosis enters the proteomics scene: new answers to old questions.Proteomics. 2006; 6: 4084-4099Crossref PubMed Scopus (39) Google Scholar). In general, there is a weak correlation between genotype and phenotype (3Sermet-Gaudelus I. Dechaux M. Vallee B. Fajac A. Girodon E. Nguyen-Khoa T. Marianovski R. Hurbain I. Bresson J.L. Lenoir G. Chloride transport in nasal ciliated cells of cystic fibrosis heterozygotes.Am. J. Respir. Crit. Care Med. 2005; 171: 1026-1031Crossref PubMed Scopus (34) Google Scholar), which strongly suggests either the existence of modifier genes or a strong influence of epigenetic factors, such as environment and methylation/acetylation of DNA and/or proteins. Modifier genes have been the subject of an extensive body of research in the last few years (recently reviewed in Refs. 4Accurso F.J. Sontag M.K. Gene modifiers in cystic fibrosis.J. Clin. Invest. 2008; 118: 839-841Crossref PubMed Scopus (25) Google Scholar and 5Collaco J.M. Cutting G.R. Update on gene modifiers in cystic fibrosis.Curr. Opin. Pulm. Med. 2008; 14: 559-566Crossref PubMed Scopus (102) Google Scholar). Exploration of epigenetic factors in CF is in its beginning stage (6Shuto T. Furuta T. Oba M. Xu H. Li J.D. Cheung J. Gruenert D.C. Uehara A. Suico M.A. Okiyoneda T. Promoter hypomethylation of Toll-like receptor-2 gene is associated with increased proinflammatory response toward bacterial peptidoglycan in cystic fibrosis bronchial epithelial cells.FASEB J. 2006; 20: 782-784Crossref PubMed Scopus (92) Google Scholar). This limited correlation makes it difficult to estimate the prognosis and evolution of the disease, and to optimize treatments and prophylactic actions. The search for predictive or prognostic biomarkers has become capital in the CF field. Biomarkers obtained by rapid, noninvasive methods could be used in the earlier detection of CF exacerbations or as prognostic indicators. Intense proteomics research to unveil protein markers has resulted in finding associations between myeloperoxidase, interleukin (IL)-8, cleaved α-antitrypsin, and S100A8 (CF antigen) in sputum and the frequency of pulmonary exacerbations (7Sloane A.J. Lindner R.A. Prasad S.S. Sebastian L.T. Pedersen S.K. Robinson M. Bye P.T. Nielson D.W. Harry J.L. Proteomic analysis of sputum from adults and children with cystic fibrosis and from control subjects.Am. J. Respir. Crit. Care Med. 2005; 172: 1416-1426Crossref PubMed Scopus (76) Google Scholar) or directly in the presence of CFTR mutations (8Gray R.D. MacGregor G. Noble D. Imrie M. Dewar M. Boyd A.C. Innes J.A. Porteous D.J. Greening A.P. Sputum proteomics in inflammatory and suppurative respiratory diseases.Am. J. Respir. Crit. Care Med. 2008; 178: 444-452Crossref PubMed Scopus (146) Google Scholar, 9Wilkinson M.M. Busuttil A. Hayward C. Brock D.J. Dorin J.R. Van Heyningen V. Expression pattern of two related cystic fibrosis-associated calcium-binding proteins in normal and abnormal tissues.J. Cell Sci. 1988; 91: 221-230PubMed Google Scholar), in addition to proteomic signatures in serum or corresponding to known inflammation markers (10Srivastava M. Eidelman O. Jozwik C. Paweletz C. Huang W. Zeitlin P.L. Pollard H.B. Serum proteomic signature for cystic fibrosis using an antibody microarray platform.Mol. Genet. Metab. 2006; 87: 303-310Crossref PubMed Scopus (65) Google Scholar) or other proteins differentially expressed in target tissues of CF before the appearance of any sign of the disease, like annexin-1 (11Bensalem N. Ventura A.P. Vallee B. Lipecka J. Tondelier D. Davezac N. Dos Santos A. Perretti M. Fajac A. Sermet-Gaudelus I. Down-regulation of the anti-inflammatory protein annexin A1 in cystic fibrosis knock-out mice and patients.Mol. Cell. Proteomics. 2005; 4: 1591-1601Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), cytosolic phospholipase A2α (11Bensalem N. Ventura A.P. Vallee B. Lipecka J. Tondelier D. Davezac N. Dos Santos A. Perretti M. Fajac A. Sermet-Gaudelus I. Down-regulation of the anti-inflammatory protein annexin A1 in cystic fibrosis knock-out mice and patients.Mol. Cell. Proteomics. 2005; 4: 1591-1601Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), cytokeratins 8 and 18 (12Davezac N. Tondelier D. Lipecka J. Fanen P. Demaugre F. Debski J. Dadlez M. Schrattenholz A. Cahill M.A. Edelman A. Global proteomic approach unmasks involvement of keratins 8 and 18 in the delivery of cystic fibrosis transmembrane conductance regulator (CFTR)/deltaF508-CFTR to the plasma membrane.Proteomics. 2004; 4: 3833-3844Crossref PubMed Scopus (48) Google Scholar), and peroxiredoxin 6 (13Trudel S. Kelly M. Fritsch J. Nguyen-Khoa T. Therond P. Couturier M. Dadlez M. Debski J. Touqui L. Vallee B. Peroxiredoxin 6 fails to limit phospholipid peroxidation in lung from cftr-knockout mice subjected to oxidative challenge.PLoS ONE. 2009; 4: e6075Crossref PubMed Scopus (27) Google Scholar, 14Gaggar A. Jackson P.L. Noerager B.D. O'Reilly P.J. McQuaid D.B. Rowe S.M. Clancy J.P. Blalock J.E. A novel proteolytic cascade generates an extracellular matrix-derived chemoattractant in chronic neutrophilic inflammation.J. Immunol. 2008; 180: 5662-5669Crossref PubMed Scopus (262) Google Scholar). More recently, sputum proteomics has revealed that a proline-glycine-proline (PGP) peptide, an extracellular matrix-derived neutrophil attractant, is a marker of inflammatory exacerbation in CF (14Gaggar A. Jackson P.L. Noerager B.D. O'Reilly P.J. McQuaid D.B. Rowe S.M. Clancy J.P. Blalock J.E. A novel proteolytic cascade generates an extracellular matrix-derived chemoattractant in chronic neutrophilic inflammation.J. Immunol. 2008; 180: 5662-5669Crossref PubMed Scopus (262) Google Scholar). In a recent report, a SELDI-based screening of serum and nasal cells from patients with CF and other respiratory diseases suggested that combinations of peptide ions studied by multivariate analysis have a better predictive value than individual species (15Gomes-Alves P. Imrie M. Gray R.D. Nogueira P. Ciordia S. Pacheco P. Azevedo P. Lopes C. de Almeida A.B. Guardiano M. SELDI-TOF biomarker signatures for cystic fibrosis, asthma and chronic obstructive pulmonary disease.Clin. Biochem. 2010; 43: 168-177Crossref PubMed Scopus (21) Google Scholar). In addition to proteins and peptides, smaller molecules like metabolites and particularly lipids may represent an interesting target in the biomarker search. Several pioneering metabolomic profiling studies in CF have been reported very recently. The first revealed the presence of a high content of metabolites, such as amino acids and lactate, as markers of inflammation in broncho-alveolar fluid from CF patients (16Wolak J.E. Esther Jr, C.R. O'Connell T.M. Metabolomic analysis of bronchoalveolar lavage fluid from cystic fibrosis patients.Biomarkers. 2009; 14: 55-60Crossref PubMed Scopus (92) Google Scholar). The second unveiled modifications in purinergic signaling, glucose metabolism, and the response to oxidative stress in CF cells (17Wetmore D.R. Joseloff E. Pilewski J. Lee D.P. Lawton K.A. Mitchell M.W. Milburn M.V. Ryals J.A. Guo L. Metabolomic profiling reveals biochemical pathways and biomarkers associated with pathogenesis in cystic fibrosis cells.J. Biol. Chem. 2010; 285: 30516-30522Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The third presented volatile organic compounds as markers of Pseudomonas aeruginosa colonization (18Robroeks C.M. van Berkel J.J. Dallinga J.W. Jobsis Q. Zimmermann L.J. Hendriks H.J. Wouters M.F. van der Grinten C.P. van de Kant K.D. van Schooten F.J. Metabolomics of volatile organic compounds in cystic fibrosis patients and controls.Pediatr. Res. 2010; 68: 75-80Crossref PubMed Scopus (132) Google Scholar). With reference to lipids, alterations in the lipid content of cells and fluids of CF patients and models have been reported since the early sixties. These alterations include a decrease in circulating essential fatty acids (19Farrell P.M. Mischler E.H. Engle M.J. Brown D.J. Lau S.M. Fatty acid abnormalities in cystic fibrosis.Pediatr. Res. 1985; 19: 104-109Crossref PubMed Scopus (136) Google Scholar, 20Roulet M. Frascarolo P. Rappaz I. Pilet M. Essential fatty acid deficiency in well nourished young cystic fibrosis patients.Eur. J. Pediatr. 1997; 156: 952-956Crossref PubMed Scopus (133) Google Scholar), phospholipids, and lysophospholipids (21Guerrera I.C. Astarita G. Jais J.P. Sands D. Nowakowska A. Colas J. Sermet-Gaudelus I. Schuerenberg M. Piomelli D. Edelman A. A novel lipidomic strategy reveals plasma phospholipid signatures associated with respiratory disease severity in cystic fibrosis patients.PLoS ONE. 2009; 4: e7735Crossref PubMed Scopus (45) Google Scholar); decreased polyunsaturated fatty acids in lung, intestine, and pancreas of mice (22Freedman S.D. Blanco P.G. Shea J.C. Alvarez J.G. Analysis of lipid abnormalities in CF mice.Methods Mol. Med. 2002; 70: 517-524PubMed Google Scholar, 23Freedman S.D. Katz M.H. Parker E.M. Laposata M. Urman M.Y. Alvarez J.G. A membrane lipid imbalance plays a role in the phenotypic expression of cystic fibrosis in cftr(−/−) mice.Proc. Natl. Acad. Sci. USA. 1999; 96: 13995-14000Crossref PubMed Scopus (258) Google Scholar) and nasal cells of patients (24Freedman S.D. Blanco P.G. Zaman M.M. Shea J.C. Ollero M. Hopper I.K. Weed D.A. Gelrud A. Regan M.M. Laposata M. Association of cystic fibrosis with abnormalities in fatty acid metabolism.N. Engl. J. Med. 2004; 350: 560-569Crossref PubMed Scopus (337) Google Scholar), mostly associated with an imbalanced n-3/n-6 ratio; and decreased linoleic acid (19Farrell P.M. Mischler E.H. Engle M.J. Brown D.J. Lau S.M. Fatty acid abnormalities in cystic fibrosis.Pediatr. Res. 1985; 19: 104-109Crossref PubMed Scopus (136) Google Scholar, 25Ollero M. Laposata M. Zaman M.M. Blanco P.G. Andersson C. Zeind J. Urman Y. Kent G. Alvarez J.G. Freedman S.D. Evidence of increased flux to n-6 docosapentaenoic acid in phospholipids of pancreas from cftr−/− knockout mice.Metabolism. 2006; 55: 1192-1200Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) and lipoxin A4 in lung exudates from patients (26Karp C.L. Flick L.M. Park K.W. Softic S. Greer T.M. Keledjian R. Yang R. Uddin J. Guggino W.B. Atabani S.F. Defective lipoxin-mediated anti-inflammatory activity in the cystic fibrosis airway.Nat. Immunol. 2004; 5: 388-392Crossref PubMed Scopus (306) Google Scholar). These alterations have been attributed largely to malabsorption (19Farrell P.M. Mischler E.H. Engle M.J. Brown D.J. Lau S.M. Fatty acid abnormalities in cystic fibrosis.Pediatr. Res. 1985; 19: 104-109Crossref PubMed Scopus (136) Google Scholar), increased flux through the n-6 biosynthetic pathway (25Ollero M. Laposata M. Zaman M.M. Blanco P.G. Andersson C. Zeind J. Urman Y. Kent G. Alvarez J.G. Freedman S.D. Evidence of increased flux to n-6 docosapentaenoic acid in phospholipids of pancreas from cftr−/− knockout mice.Metabolism. 2006; 55: 1192-1200Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 27Andersson C. Al-Turkmani M.R. Savaille J.E. Alturkmani R. Katrangi W. Cluette-Brown J.E. Zaman M.M. Laposata M. Freedman S.D. Cell culture models demonstrate that CFTR dysfunction leads to defective fatty acid composition and metabolism.J. Lipid Res. 2008; 49: 1692-1700Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 28Strandvik B. Gronowitz E. Enlund F. Martinsson T. Wahlstrom J. Essential fatty acid deficiency in relation to genotype in patients with cystic fibrosis.J. Pediatr. 2001; 139: 650-655Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), and an alteration in the methionine-homocysteine cycle (29Innis S.M. Davidson A.G. Cystic fibrosis and nutrition: linking phospholipids and essential fatty acids with thiol metabolism.Annu. Rev. Nutr. 2008; 28: 55-72Crossref PubMed Scopus (37) Google Scholar, 30Innis S.M. Hasman D. Evidence of choline depletion and reduced betaine and dimethylglycine with increased homocysteine in plasma of children with cystic fibrosis.J. Nutr. 2006; 136: 2226-2231Crossref PubMed Scopus (44) Google Scholar). As in protein biomarker research, we have recently evoked the usefulness of multivariate analysis in lipidomics (31Brulet M. Seyer A. Edelman A. Brunelle A. Fritsch J. Ollero M. Laprevote O. Lipid mapping of colonic mucosa by cluster TOF-SIMS imaging and multivariate analysis in cftr knockout mice.J. Lipid Res. 2010; 51: 3034-3045Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). In a recent paper, we have shown altered phospholipid plasma content associated with both the onset and severity status of the disease (21Guerrera I.C. Astarita G. Jais J.P. Sands D. Nowakowska A. Colas J. Sermet-Gaudelus I. Schuerenberg M. Piomelli D. Edelman A. A novel lipidomic strategy reveals plasma phospholipid signatures associated with respiratory disease severity in cystic fibrosis patients.PLoS ONE. 2009; 4: e7735Crossref PubMed Scopus (45) Google Scholar). In the present work, we have addressed the same question by analyzing the plasma lipid composition of CF patients in a cohort composed exclusively of F508del homozygotes who shared the same treatment modalities and follow-up from one clinical center. On the basis of previous reports, in this study we have covered the main lipid classes by targeting our analysis on phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) species, as well as on free fatty acids, cholesterol, cholesterol esters, triglycerides, and ceramides. We have also addressed for the first time in CF the analysis of fatty acyl amides. We have compared these lipid signatures at one time point with two clinical parameters. The first parameter was the forced respiratory volume in 1 s (FEV1), considered today the most reliable marker of respiratory function and disease severity in CF. The second parameter was chronic infection by Pseudomonas aeruginosa, the most common bacteria in CF patients, a marker of the inflammation-infection scenario, and one of the main prognostic indicators of worsening in lung disease. Both parameters were measured at two time points, which represent a longitudinal study of disease evolution and a cross-sectional lipid screening. As a result, we show the correlation of the plasma content of a series of lipid species with relevant clinical parameters at both time points. This suggests a potential prognostic value for some of those lipid signatures in relation to the progression of CF disease. All protocols were approved by the Ile-de-France II ethical committee. All patients involved in the study or their parents or legal guardians signed a written consent form. Blood plasma samples were collected from CF patients at Hôpital Necker Enfants Malades (Paris, France) using a standard clinical protocol. All patients were at a good nutritional status and out of exacerbation period at the time of collection. All patients were receiving vitamins A, D, E, and K supplements as well as pancreatic enzymes. Sixteen patients were receiving inhaled corticoids. Blood collection was done at fasting. The analysis was performed on samples from 44 patients (description of patients is presented in Table 1). Sample collection was carried out with appropriate ethical committee approval. Samples were collected in VACUETTE® EDTA tubes K3E/EDTA K3 (Greiner Bio-One, Kremsmünster, Austria) and centrifuged at 2800 g for 15 min at 4°C. Plasma was separated and dispensed into 100 µl aliquots so that each aliquot was subjected to a single freezing-thawing cycle. Plasma samples were frozen in liquid nitrogen and stored at −80°C.TABLE 1Description of patientsPatient CodeAge at Inclusion(years)FEV1%(t = 0)FEV1%(t = 3)Chronic Colonization(t = 0)Chronic Colonization(t = 3)Infection Evolution Group1109974NoNoA2179760NoND3196770YesNoD4192427NoYesB53NDNDNoND64ND40NoYesB7177954NoNoA8128579NoYesB9143530YesND10175091YesYesC11710088YesYesC1279883NoNoA1385856YesYesC141NDNDYesYesC159ND39NoYesB16183554YesYesC174164NDNoND18136063YesND193320NDYesND20143424YesYesC212NDNDNoYesB2298872NoNoA23109495NoYesB24177184NoYesB259NDNDYesND262831NDYesND272NDNDNoNoA281NDNDNoYesB298ND75NoND301556NDNoNoA311NDNDNoNoA32135440NoYesB332NDNDNoND345NDNDNoNoA35153222NoYesB3679989NoNoA37810085NoYesB385ND53NoYesB3999481NoYesB4018ND49YesYesC41185450YesYesC42137256YesYesC4316ND62NoNoA4488682NoNoAND, not determined. Open table in a new tab ND, not determined. Patients were chosen according to their CFTR mutation genotype. All patients were homozygous for F508del. Pulmonary function test results (FEV1) were expressed as the percentage of the predicted value (32Knudson R.J. Lebowitz M.D. Holberg C.J. Burrows B. Changes in the normal maximal expiratory flow-volume curve with growth and aging.Am. Rev. Respir. Dis. 1983; 127: 725-734PubMed Google Scholar). FEV1 could only be recorded in 29 patients at the beginning of the study and in 31 patients three years later. In most cases, this was due to the short age of patients. Chronic colonization with Pseudomonas aeruginosa (defined by at least three positive sputum cultures within three months) was recorded for each patient at the beginning of the study and for 34 patients three years later. Aliquots of 100 µl of plasma were subjected to organic extraction by addition of six volumes of chloroform-methanol 2:1 (v/v) containing a mixture of internal standards (all from Avanti Polar Lipids, Alabaster, AL, unless stated otherwise); namely, heptadecanoic acid (Nu-Chek Prep, Elysian, MN), d8-arachidonic acid (Cayman Chemicals, Ann Arbor, MI), heptadecenoylethanolamide (synthesized as previously reported (33Astarita G. Ahmed F. Piomelli D. Identification of biosynthetic precursors for the endocannabinoid anandamide in the rat brain.J. Lipid Res. 2008; 49: 48-57Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar)), trinonadecenoin (Nu-Chek Prep), dinonadienoyl-sn-glycerol (Nu-Chek Prep), monoheptadecanoyl-sn-glycerol (Nu-Chek Prep), d8-2-arachidonoyl-sn-glycerol (Cayman Chemicals), 1,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine, 1,2-diheptadecanoyl-sn-glycero-3-phosphoglycerol, 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine, 1,2-diheptadecanoyl-sn-glycero-3-phosphoserine, 1,2-dipalmitoyl-sn-glycero-3-phosphoinositol, N-lauroyl-ceramide, N-lauroyl-sphingomyelin, and d7-cholesterol. LC-MS analyses were performed using an Agilent 1200-LC system coupled to a 1946D or Ion-Trap XCT detector interfaced with ESI or APCI (Agilent Technologies, Wilmington, DE). To separate lipids containing one fatty acyl group, a reversed-phase C-18 column packed with conventional porous silica particles of small spherical diameter (Zorbax XDB Eclipse C-18 column from Agilent Technologies, 50 × 4.6 mm id, 1.8 µm particle size, 80 Å pore size) was used. Fatty acyl species were separated by a binary liquid chromatography method. Mobile phase A consisted of methanol containing 0.25% acetic acid and 5 mM ammonium acetate, and mobile phase B corresponded to water containing 0.25% acetic acid and 5 mM ammonium acetate. Separation was performed using a linear gradient from 90% A to 100% B in 2.5 min at a flow rate of 1.5 ml/min with column temperature at 40°C. ESI was set in the negative mode at −4.0 kV capillary voltage and 100V fragmentor voltage. N2 was used as drying gas at a flow rate of 12 l/min with temperature of 350°C and nebulizer pressure of 60 psi. Fatty acids were analyzed monitoring the mass-to-charge ratio (m/z) of the deprotonated molecular ions [M-H]− in selected-ion monitoring mode. To separate glycerolipids, glycerophospholipids, sphingolipids, and sterol lipids, a reversed-phase Poroshell 300SB C-18 column (2.1 × 75 mm id, coating layer of 0.25 µm on total particle diameter of 5 µm, 300 Å pore size; Agilent Technologies) was used. A linear gradient was applied from 85% A to 100% B in 5 min (75% A to 100% B in 4 min in the case of sterol lipids) at a flow rate of 1.0 ml/min with column temperature set at 50°C. MS detection was performed both in the positive and negative ionization modes. The capillary voltage was set at −4.0 kV, and the skimmer voltage at 40V. N2 was used as drying gas at a flow rate of 10 l/min with temperature of 350°C and nebulizer pressure of 60 psi. Helium was used as collision gas, and fragmentation amplitude was set at 1.2V. For sterol lipids, APCI was set in positive mode, drying gas at 350°C, flow rate of 8 l/min, nebulizer gas pressure at 30 psi, vaporizer temperature at 475°C. Capillary voltage was 300V with the corona current at 5 μA. Lipids were identified by comparing their LC retention times and MSn fragmentation patterns with those of authentic standards as previously described (33Astarita G. Ahmed F. Piomelli D. Identification of biosynthetic precursors for the endocannabinoid anandamide in the rat brain.J. Lipid Res. 2008; 49: 48-57Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 34Astarita G. Piomelli D. Lipidomic analysis of endocannabinoid metabolism in biological samples.J Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2009; 877: 2755-2767Crossref PubMed Scopus (76) Google Scholar). Because of the coexistence of multiple isobaric and isomeric species, not every individual molecular species of glycerophospholipids could be quantified. For example, the isobars PC(16:1/16:1) and PC(14:0/18:2) were quantified together and expressed as PC(32:2). Detection and analysis were controlled by Agilent/Bruker Daltonics software version 5.2. Signatures were compared with clinical parameters recorded at the time of sample collection (t = 0) and three years later (t = 3). Patients 2 and 15 showed abnormally high values for most free fatty acids (about 10-fold). Where indicated, these patients were excluded from the analysis. For some patients, the analytical data corresponding to some lipids are missing due to technical problems. Also, some clinical parameters could not be recorded at both time points for some patients. The number of data available is indicated for each test. Data are generally presented as means ± SEM. Statistical analysis included univariate and multivariate analyses. All variables showed normal distributions, and parametric tests were chosen. Among univariate analyses, Student's t-test was used to compare two groups of patients, ANOVA to compare more than two groups, and Pearson correlation for comparison of numeric variables within the cohort. Multivariate analysis included multiple regression for numeric dependent variables, logistic regression for nominal dependent variables, and principal component analysis (PCA). Analyses were performed with XLStat, PopTools, and GraphPad InStat software. On the basis of our previous results (21Guerrera I.C. Astarita G. Jais J.P. Sands D. Nowakowska A. Colas J. Sermet-Gaudelus I. Schuerenberg M. Piomelli D. Edelman A. A novel lipidomic strategy reveals plasma phospholipid signatures associated with respiratory disease severity in cystic fibrosis patients.PLoS ONE. 2009; 4: e7735Crossref PubMed Scopus (45) Google Scholar, 24Freedman S.D. Blanco P.G. Zaman M.M. Shea J.C. Ollero M. Hopper I.K. Weed D.A. Gelrud A. Regan M.M. Laposata M. Association of cystic fibrosis with abnormalities in fatty acid metabolism.N. Engl. J. Med. 2004; 350: 560-569Crossref PubMed Scopus (337) Google Scholar) and reports by others (35Guilbault C. De Sanctis J.B. Wojewodka G. Saeed Z. Lachance C. Skinner T.A. Vilela R.M. Kubow S. Lands L.C. Hajduch M. Fenretinide corrects newly found ceramide deficiency in cystic fibrosis.Am. J. Respir. Cell Mol. Biol. 2008; 38: 47-56Crossref PubMed Scopus (89) Google Scholar, 36Teichgraber V. Ulrich M. Endlich N. Riethmuller J. Wilker B. De Oliveira-Munding C.C. van Heeckeren A.M. Barr M.L. von Kurthy G. Schmid K.W. Ceramide accumulation mediates inflammation, cell death and infection susceptibility in cystic fibrosis.Nat. Med. 2008; 14: 382-391Crossref PubMed Scopus (458) Google Scholar, 37White N.M. Jiang D. Burgess J.D. Bederman I.R. Previs S.F. Kelley T.J. Altered cholesterol homeostasis in cultured and in vivo models of cystic fibrosis.Am. J. Physiol. Lung Cell. Mol. Physiol. 2007; 292: L476-L486Crossref PubMed Scopus (71) Google Scholar), we first focused our lipidomic screening on 50 lipid molecules or entities. After multiple Pearson correlation analysis of the lipid values at t = 0 with FEV1 measured at sampling time, a series of lipid signatures were found correlated with this respiratory functional parameter (n = 29, p < 0.05). These lipid signatures corresponded to free fatty acids, including saturated, mono-, and polyunsaturated species (Table 2).TABLE 2Lipid species correlated significantly with FEV1 values obtained at t = 0Lipid SpeciesrpC16:1n-70.39190.0432C16:00.47020.0133C18:1n-90.38730.0459C18:00.48570.0102C18:3n-30.38170.0494C18:2n-60.37320.0461C20:3n-60.52080.0059C20:3n-90.57350.0018C22:5n-60.41480.0314C22:5n-30.56330.0022C20:5n-30.40350.0369C22:6n-3aPatient 2 was excluded due to abnormally high values.0.37440.0497The analysis was performed by linear regression (p < 0.05).a Patient 2 was excluded due to abnormally high values. Open table in a new tab The analysis was performed by linear regression (p < 0.05). Interestingly, selected PUFA were significantly correlated with FEV1 at t = 3 (n = 31), which confers them a predictive potential on this parameter. This included C20:5n-3, C22:5n-3, and C22:6n-3, known as anti-inflammatory molecules, and C20:3n-9, the endpoint of the n-9 series. Also, two PC species [PC(36:4) and PC(32:2)] were also positively correlated with FEV1 at t = 3 (n = 31). These lipids and their respective correlation values are presented in Table 3. In addition, the desaturation index, defined by the C16:1/C16:0 ratio, was also positively correlated with FEV1 at t = 3 (n = 31, r = 0.4540, p = 0.0117). Fig. 1 shows
Referência(s)