Cholesteryl Esters Are Elevated in the Lipid Fraction of Bronchoalveolar Lavage Fluid Collected from Pediatric Cystic Fibrosis Patients

<div><p>Background</p><p>Host-derived lipids including cholesteryl esters (CEs) such as cholesteryl linoleate have emerged as important antibacterial effectors of innate immunity in the airways and cholesteryl linoleate has been found elevated in the context of inflammation. Cystic fibrosis (CF) patients suffer from chronic infection and severe inflammation in the airways. Here, we identified and quantified CEs in bronchoalveolar lavage fluid (BALF) from CF patients and non-CF disease controls, and tested whether CE concentrations are linked to the disease.</p><p>Materials and Methods</p><p>CEs in BALF from 6 pediatric subjects with CF and 7 pediatric subjects with non-CF chronic lung disease were quantified by mass spectral analysis using liquid chromatography coupled with tandem mass spectrometry and multiple reaction monitoring. BALFs were also examined for total lipid, total protein, albumin, and, as a marker for inflammation, human neutrophil peptide (HNP) 1–3 concentrations. Statistical analysis was conducted after log 10 transformation of the data.</p><p>Results</p><p>Total lipid/protein ratio was reduced in CF BALF (<i>p</i> = 0.018) but the concentrations of CEs, including cholesteryl linoleate, were elevated in the total lipid fraction in CF BALF compared to non-CF disease controls (<i>p</i> < 0.050). In addition, the concentrations of CEs and HNP1-3 correlated with one another (p < 0.050).</p><p>Conclusions</p><p>The data suggests that the lipid composition of BALF is altered in CF with less total lipid relative to protein but with increased CE concentrations in the lipid fraction, likely contributed by inflammation. Future longitudinal studies may reveal the suitability of CEs as a novel biomarker for CF disease activity which may provide new information on the lipid mediated pathophysiology of the disease.</p></div

Correlation between Cholesteryl Esters and Markers of Inflammation.

<p>^a For each cholesteryl ester the fatty acid residue esterified to cholesterol is given. Correlation was calculated with log transformed data (log₁₀ [1+data]).</p><p>*Correlation is significant at the 0.05 level (2-tailed).</p><p>**Correlation is significant at the 0.01 level (2-tailed). P-values indicating statistical significance are shown in bold. CL: cholesteryl linoleate; CA: cholesteryl arachidonate.</p><p>Correlation between Cholesteryl Esters and Markers of Inflammation.</p

Description of Study Population and BALF Characteristics.

<p>^a Chronic lung disease;</p><p>^b Interstitial lung disease;</p><p>^c Subglottic;</p><p>^d Neuroendocrine hyperplasia of infancy;</p><p>^e Negative indicates no growth or normal microbiota isolated, positive indicates isolation of probable pathogens;</p><p>^f CF genotype is given.</p><p>Description of Study Population and BALF Characteristics.</p

Total Lipid and Protein Analysis of BALF.

<p>^a RFU: relative fluorescence units;</p><p>^b ND: not detectable.</p><p>Total Lipid and Protein Analysis of BALF.</p

CE fraction of total lipid.

<p>CEs are identified by the fatty acid species (number of carbons: number of unsaturated bonds) attached to the cholesterol molecule (C). Individual CE concentrations are given for each molecular species identified as well as for cholesteryl linoleate (C18:2) and cholesteryl arachidonate (C20:4) combined and all CEs combined. Shown are the means ± S.E.M of log transformed data with n = 7 for Non-CF and n = 6 for CF. * indicates a <i>p</i> value of < 0.050 in one-tailed <i>t</i>-test.</p

CE concentrations in BALF collected from non-CF disease control subjects (top) and CF patients (bottom).

<p>CEs are identified by the fatty acid species (number of carbons: number of unsaturated bonds) attached to the cholesterol molecule (C). Individual CE concentrations are given for each molecular species identified as well as for cholesteryl linoleate (C18:2) and cholesteryl arachidonate (C20:4) combined and all CEs combined.</p

Quantification of HNP1-3 by Western immunoblot.

<p>HNP2 peptide standard and bronchoalveolar lavage fluid (BALF) samples were separated by 4–20% SDS PAGE, blotted onto PVDF-PSQ membranes, probed with polyclonal rabbit antibodies against HNP1-3, and antibody binding was visualized with goat-anti rabbit antibodies conjugated to alkaline phosphatase and NBT/BCIP substrate. HNP1-3 differ by one amino acid only and co-migrate in this gel system. Per lane, the equivalent of the following BALF volumes were loaded: for Non-CF samples, 1: 20 μL, 3: 40 μL, 4: 40 μL, 6: 20 μL, 9: 40 μL, 10: 20 μL, 12: 20 μL; for CF samples, 2: 6 μL, 5: 6 μL, 7: 6 μL, 8: 20 μL, 11: 6 μL, 13: 6 μL.</p

Cholesteryl Esters Described in this Study.

<p>^a Carbon number: number of double bonds;</p><p>^b in stable ¹³C1 isotope form.</p><p>Cholesteryl Esters Described in this Study.</p