Long-chain Acyl-CoA Dehydrogenase Deficiency as a Cause of Pulmonary Surfactant Dysfunction*

Background: The contribution of long-chain acyl-CoA dehydrogenase (LCAD) to human fatty acid oxidation is not understood. Results: LCAD localizes to lung alveolar type II cells, which produce pulmonary surfactant; LCAD-deficient mice have surfactant dysfunction. Conclusion: LCAD is important for lung energy metabolism and lung function. Significance: LCAD may play a role in human lung disease and unexplained sudden infant death. Long-chain acyl-CoA dehydrogenase (LCAD) is a mitochondrial fatty acid oxidation enzyme whose expression in humans is low or absent in organs known to utilize fatty acids for energy such as heart, muscle, and liver. This study demonstrates localization of LCAD to human alveolar type II pneumocytes, which synthesize and secrete pulmonary surfactant. The physiological role of LCAD and the fatty acid oxidation pathway in lung was subsequently studied using LCAD knock-out mice. Lung fatty acid oxidation was reduced in LCAD−/− mice. LCAD−/− mice demonstrated reduced pulmonary compliance, but histological examination of lung tissue revealed no obvious signs of inflammation or pathology. The changes in lung mechanics were found to be due to pulmonary surfactant dysfunction. Large aggregate surfactant isolated from LCAD−/− mouse lavage fluid had significantly reduced phospholipid content as well as alterations in the acyl chain composition of phosphatidylcholine and phosphatidylglycerol. LCAD−/− surfactant demonstrated functional abnormalities when subjected to dynamic compression-expansion cycling on a constrained drop surfactometer. Serum albumin, which has been shown to degrade and inactivate pulmonary surfactant, was significantly increased in LCAD−/− lavage fluid, suggesting increased epithelial permeability. Finally, we identified two cases of sudden unexplained infant death where no lung LCAD antigen was detectable. Both infants were homozygous for an amino acid changing polymorphism (K333Q). These findings for the first time identify the fatty acid oxidation pathway and LCAD in particular as factors contributing to the pathophysiology of pulmonary disease.

causing mutations have been identified for LCAD, and its role in human metabolism remains obscure.
In vitro, the substrate specificity of LCAD overlaps with that of VLCAD and ACAD9. All three enzymes have strong activity toward long-chain acyl-CoAs (14 -20 carbons in length) (4). Additionally, LCAD has been shown to dehydrogenate branched-chain acyl-CoA substrates (5). VLCAD expression predominates in tissues that are known to rely upon fatty acids for energy such as liver, heart, and muscle. Correspondingly, VLCAD deficiency causes life-threatening metabolic decompensation, cardiomyopathy, and muscle weakness (6,7). ACAD9 is also expressed in muscle and heart and is the only ACAD enzyme expressed at high levels in the brain and central nervous system (4). Additionally, ACAD9 interacts with complex I of the respiratory chain and may have other functions besides its ACAD activity (8,9). In contrast to VLCAD and ACAD9, LCAD expression is low or nearly absent in liver, heart, and muscle and most abundant in lung, kidney, thyroid, and prostate (4,10,11).
The presence of the LCAD protein has been confirmed in human lung using immunostaining (4). In cultured primary human alveolar type II pneumocytes (ATII), influenza infection was found to down-regulate LCAD mRNA, whereas other FAO genes were not affected (12). Similarly, based on microarray studies in lung tumors, LCAD was included in a core set of genes whose expression levels distinguish cancerous from normal tissue (13). These reports prompted us to hypothesize that LCAD may play a role in fatty acid metabolism specific to lung. In this study, we confirmed localization of LCAD to human ATII cells. ATII cells are highly specialized cells in the alveolar epithelium that synthesize and secrete pulmonary surfactant. Surfactant is a mixture of phospholipids, cholesterol, and protein that allows for decreased surface tension at the air-liquid interface of the lung, preventing alveolar collapse and allowing for normal gas exchange. We further observed surfactant deficiency and altered lung mechanics in LCAD-deficient mice. Based on these findings, it is postulated that LCAD deficiency in humans may manifest primarily as a lung disease rather than resembling other FAO disorders.

EXPERIMENTAL PROCEDURES
Human Tissues, Cell Isolation, and Cell Culture-Post-mortem human lung tissues from children who suffered unexplained deaths during the 1st year of life were acquired through the University of Maryland Tissue Bank. Primary human ATII cells and bronchial epithelial cells were isolated from adult donor lungs not suitable for transplantation as described (14). Briefly, the right middle lobe was perfused, lavaged, and instilled with elastase (Roche Diagnostics). The tissue was minced, and ATII cells were purified by centrifugation through an Optiprep gradient (Accurate Chemical Scientific, Westbury, NY) followed by negative selection with CD14-coated magnetic beads (Dynal Biotech, Oslo, Norway) and binding to IgGcoated dishes (Sigma). The resulting ATII cell cultures were then either maintained as differentiated ATII cells for 6 days as described (15) or trans-differentiated into ATI-like cells by culturing for 2 days in DMEM with 10% FBS on rat tail collagencoated plates followed by 4 days in DMEM with 5% FBS (16).
The A549 human alveolar adenocarcinoma cell line and the MLE12 transformed murine lung epithelial cell line were from ATCC, and they were cultured as described previously (17).
Animals-All breeding and experimental protocols were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. LCAD ϩ/Ϫ mice (B6.129S6-Acadl tm1Uab ) were purchased from the Mutant Mouse Regional Resource Center (University of Missouri, Columbia, MO). LCAD Ϫ/Ϫ mice were backcrossed to C57Bl/6 but became infertile. Thus, the LCAD Ϫ/Ϫ mice used here were maintained on a mixed C57Bl/6 and 129S6 background. Wild-type and homozygous mutant littermates were identified from initial heterozygous crosses and used to establish separate breeding colonies of wild-type and homozygous mutant mice. All studies used age-and sex-matched animals.
Immunoblotting of Tissue, Cell Lysates, and Lavage Fluid-Frozen lung tissue was homogenized in 50 mM Tris-HCl, pH 8.0, 2 mM EDTA, and 0.25% lubrol detergent, pH 8.0, with protease inhibitors and incubated on ice for 15 min. Cultured cells were lysed in RIPA buffer. Both tissue and cell homogenates were cleared by centrifugation and analyzed for protein concentration in triplicate using the Bradford method (Bio-Rad). Lysates were electrophoresed and transferred to nitrocellulose membranes using the Bio-Rad Criterion apparatus. Anti-LCAD, VLCAD, and ACAD9 antibodies, generated previously in our laboratory, were used as described (18). Anti-actin antibody (Santa Cruz Biotechnology, Dallas, TX) was used as a loading control. In comparisons of different cell types (Fig. 1b), both actin and GAPDH were unsuitable as loading controls due to differential expression between cell types. In this case, equal loading between lanes was verified using Ponceau S staining of the nitrocellulose membranes following transfer. For Western blots of lavage fluid, the abundance of albumin and phospholipase A 2 (PLA 2 ) per volume of fluid was determined by blotting either 10 l of fluid from each animal for albumin or, in the case of PLA 2 , by dialyzing/concentrating 800 l of lavage fluid from each animal to 10 -20 l prior to loading onto the gel. Antimouse albumin (Sigma) and anti-PLA 2 (Santa Cruz Biotechnology) were both used at 1:1000.
ACAD Enzyme Activity-Frozen tissue was homogenized in 50 mM Tris-HCl, 2 mM EDTA, pH 8.0. Lubrol detergent was added to 0.25% w/v. After 15 min of incubation on ice, the samples were centrifuged, and supernatants used for ACAD enzyme activity measurements using the anaerobic electron transfer flavoprotein (ETF) fluorescence reduction assay were as described previously (19). Briefly, tissue homogenate and pure ETF were incubated together in a sealed, degassed quartz cuvette at 32°C in a heated cuvette block, and the enzymatic reaction was started by the addition of either palmitoyl-CoA or oleoyl-CoA (Sigma) at a final concentration of 25 M. The decrease in ETF fluorescence was followed for 60 s using a Jasco fluorescence spectrophotometer. Activity was calculated with 1 milliunit of activity defined as the amount of enzyme necessary to completely reduce 1 nmol of ETF in 1 min. Final activity values were calculated as milliunits of activity/mg of total protein used in the reaction.
Analysis of Free Coenzyme-A-Freshly harvested samples of mouse brown adipose tissue, liver, and lung were weighed and snap-frozen in liquid nitrogen. The frozen tissue pieces were homogenized in perchloric acid using a Bullet Blender (Next Advance, Averill Park, NY). Following centrifugation, the supernatants were neutralized to pH 6.0 and applied to a Luna C18(2) column (Phenomenex, Torrance, CA) using a Waters HPLC. Free CoA was separated following the chromatographic conditions described by DeBuysere and Olson (20). Three concentrations of pure CoA (Sigma) were injected as standards, and the peak heights were used to generate a standard curve. The concentration of CoA in tissue samples was expressed as nanomoles/mg of tissue.
Radiolabeled Fatty Acid Oxidation-Rates of FAO were determined by tritium release using [9, 3 H-fatty acids were mixed with their corresponding unlabeled fatty acids in ethanol. The ethanol was driven off by a gentle stream of nitrogen, and the fatty acids were reconstituted in PBS/BSA to a concentration of 500 M (4ϫ stock at 10 Ci per ml). Following three 10-s cycles of sonication in a sonicating water bath, the mixtures were placed on a rotator at 37°C for 4 h to allow conjugation of the fatty acid to BSA. 3 H-Fatty acid/BSA stocks were frozen at Ϫ20°C and used within 1 month. Tissues were collected early in the light cycle into ice-cold PBS, 5 mM glucose and minced into ϳ10-mg pieces. Approximately 50 mg of each tissue was placed into wells of a 24-well tissue culture plate containing 300 l of PBS, 5 mM glucose supplemented with 1 mM carnitine and 125 M final concentration of labeled fatty acid-BSA. In some experiments, lung explants were preincubated in media supplemented with 100 M of the irreversible carnitine palmitoyltransferase-1 inhibitor etomoxir (or DMSO vehicle for control) for 30 min prior to the experiment. The plates were incubated at 37°C for 1 h. The tissue pieces and reaction buffer were then transferred into Eppendorf tubes on ice and homogenized using a Bullet Blender (Next Advance, Averill Park, NY). A 10-l portion of homogenate was set aside for protein determination (D c reagent, Bio-Rad). For [ 3 H]palmitic and [ 3 H]oleic acids, 3 H 2 O and water-soluble ␤-oxidation products (i.e. acetyl-CoA) were separated from the lung homogenate and excess labeled fatty acid substrate by chloroform/methanol extraction following the method of Bligh and Dyer (21). The aqueous layer was removed and subjected to liquid scintillation counting. For [ 3 H]octanoic acid, 3 H 2 O was separated using Dowex resin as described (18).
Pulmonary Function Testing-Respiratory mechanics were measured as described previously (22). Briefly, mice were anesthetized with pentobarbital sodium (90 mg/kg i.p.). After tracheotomy with a modified 18-gauge intravenous adapter, the mice were attached to a computer-controlled piston ventilator (FlexiVent, SCIREQ, Montreal, Quebec, Canada) and ventilated with a tidal volume of 0.2 ml and 3 cm of H 2 O positive end-expiratory pressure. Multiple linear regression was used to fit measured pressure and volume in each individual mouse to the model of linear motion of the lung (23). Model fits that resulted in a coefficient of determination Ͻ0.80 were excluded. Pressure volume analysis was completed in a series of stepwise volume inflation and deflation with a maximum lung pressure of 30 cm of H 2 O. Dose-response curves to inhaled methacholine were determined as described (22) using 0.75, 3.125, 12.5, and 50 mg/ml methacholine. Airway resistance, tissue resistance, and tissue elastance were measured. The peak response for each variable was determined, and the percent change from baseline was calculated.
Lung Histology-Three 6-week-old mice of each genotype were sacrificed by pentobarbital overdose followed by exsanguination through the renal artery. The lungs were inflated with 10% neutral-buffered formalin (Sigma) at a pressure of 25 cm of H 2 O for 10 min, removed from the animal, and placed in fresh 10% neutral-buffered formalin for 24 h before processing and paraffin embedding. H&E-stained 5-m sections were scored by a pathologist blind to the genotype of the tissues.
Bronchoalveolar Lavage Fluid Collection and Analysis-All lavage fluids analyzed for these studies were collected from nonventilated 5-6-week-old mice. Mice were anesthetized and tracheotomized as described above. Bronchoalveolar lavage was performed using three 1-ml aliquots of 0.9% NaCl. The amount of saline recovered was carefully measured and then centrifuged at 300 ϫ g for 10 min to pellet cells, which were immediately resuspended in PBS, centrifuged onto glass slides, and stained for differential counting. The cell-free lavage supernatants were removed and either frozen at Ϫ80°C or, for some experiments, subjected to centrifugation at 40,000 ϫ g for 15 min to pellet large aggregate surfactant, which was used for constrained drop surfactometry and phospholipid analyses. Within 1 month, lavage supernatants were thawed and analyzed for protein concentration (Bio-Rad), and phosphatidylcholine (PC) content using thin layer chromatography followed by the phosphorus assay as described (24,25). For some experiments, large aggregate surfactant pellets were resuspended in 200 l of saline and extracted with chloroform/methanol, and the organic layer was collected and used for the phosphorus assay.
Constrained Drop Surfactometry-The constrained drop surfactometer is a newly developed droplet-based surface tensiometer (26 -28). Large aggregate surfactant pellets, obtained as described above, were gently dispersed into 0.9% NaCl, 1.5 mM CaCl 2 , and 2.5 mM HEPES, pH 7.0, with a final phospholipid concentration of ϳ1.0 mg/ml. An ϳ10-l droplet was dispensed onto the drop holder. After drop formation, the surface tension was recorded and found to quickly decrease to an equilibrium value of ϳ22-25 millinewton/m. Once the equilibrium was established, the droplet was compressed and expanded at a rate of 3 s per cycle with a compression ratio controlled to be less than 50% of the initial surface area. At least five compression-expansion cycles were studied for each droplet. As with surfactant cycling in captive bubble surfactometers, the cycles became repeatable after the first cycle and were quantified with the minimum surface tension (␥ min ), maximum surface tension (␥ max ), and compression ratio required to reach ␥ min (CR min ) (29,30). The entire measurements were conducted under physiological conditions in an environmentally controlled chamber. Drop images were taken at a rate of 10 frames/s. The surface tension, surface area, and drop volume were determined with axisymmetric drop shape analysis (31).

LC/MS Analysis of Phospholipid Molecular Species-Lipids
were extracted using the Folch procedure (32), and the phospholipid classes were separated by high performance thin layer chromatography as described (33). The PC and phosphatidylglycerol (PG) classes were recovered, and lipid phosphorus was determined by a micro-method (34). LC/MS was performed using the Dionex Ultimate TM 3000 HPLC system coupled online to a linear ion trap mass spectrometer (LXQ Thermo-Fisher) as described (33).
Incorporation of Radiolabeled Fatty Acid and Choline into Phospholipids-Lung explants were collected and incubated with [ 14 C]palmitate/BSA (125 M) as described above but for 2 h. The tissue pieces were washed vigorously four times in cold PBS to remove excess labeled fatty acid/BSA, homogenized, and extracted by the method of Bligh and Dyer (21). The aqueous layer was discarded, and the organic layer was dried under a gently stream of nitrogen. The organic layer was reconstituted in 1 ml of chloroform, and phospholipids were separated from neutral lipids using disposable 50-mg silica SPE columns exactly as described (35). The eluates were reduced in volume to ϳ100 l by a gentle stream of nitrogen and then subjected to liquid scintillation counting. Phospholipase A 2 Activity-PLA 2 activity was measured in cell-free lavage fluid using a plate-based assay kit from Cayman Chemical (Ann Arbor, MI) following the manufacturer's instructions.
LCAD Gene Sequencing and Quantitative PCR-Genomic DNA and total RNA were isolated from frozen lung tissue using commercial kits (Qiagen, Germantown, MD). Genomic DNA was used as template for PCR using primer sets flanking each exon. PCR products were gel-isolated, purified, and subjected to sequencing. To quantify LCAD mRNA, total RNA was used for reverse transcription followed by quantitative PCR with an Applied Biosystems 7300 Real Time PCR System and SYBR Green (Applied Biosystems, Carlsbad, CA).
Statistical Analyses-Results are given as the mean Ϯ S.D. Statistical analyses represent two-tailed Student's t tests, and null hypotheses were rejected at 0.05.
Ethical Approvals-The use of animals and human tissues was approved by the Institutional Animal Care and Use Committee and Institutional Review Board, respectively, at the University of Pittsburgh.

LCAD Expression in Human Lung Localizes to the Alveolar
Epithelium-To extend a previous observation of LCAD mRNA in human lung (4), we investigated the presence of the LCAD protein in human lung biopsies and various lung-derived primary cells. Western blotting readily detected LCAD in human post-mortem lung tissue at levels comparable with wild-type mouse lung (Fig. 1a). Next, Western blotting was used to investigate LCAD expression in several major lung cell types. LCAD antigen was not detected in primary human lung microvascular endothelial cells or in primary human lung fibroblasts (data not shown) but was robustly expressed in primary human ATII cells (Fig. 1b). LCAD is clearly enriched in ATII cells as evidenced by Western blotting equal amounts of ATII cell lysate versus total lung tissue homogenate from the same individuals. Interestingly, LCAD is not expressed in A549 human lung adenocarcinoma cells, which are often used as a model of ATII cells, but is expressed in the murine lung epithelial cell line MLE12 (Fig. 1b). In contrast to isolated ATII cells, clear detection of LCAD in lung tissue homogenates requires 10 times higher protein loading (50 g in Fig. 1a versus 5 g in b). Moreover, primary human bronchial epithelial cells showed weak LCAD expression visualized only upon extended exposure to film indicating that not all lung epithelial cell types express LCAD equally (Fig. 1b, middle panel). ATI cells, which are morphologically and functionally distinct from ATII cells but are thought to arise from ATII cells (16), are difficult to isolate from human lungs. However, ATI-like cells can be differentiated from ATII cells in culture, as evidenced by changes in morphology and the attainment of ATI marker proteins such as caveolin-1 (Fig. 1c) (16). In this trans-differentiation model, we observed that LCAD expression is reduced by about half in ATII cells after 6 days of culture (Fig. 1d). LCAD expression is approximately equal between ATII cells cultured for 6 days and the same cells trans-differentiated into ATI-like cells (Fig. 1d). Together, these data indicate that LCAD localizes specifically to alveolar epithelial cells.
Lung Tissue Oxidizes Fatty Acids at a High Rate-We next established that the mitochondrial FAO pathway is active in mouse lung using liver as a control tissue. Total ACAD activity toward palmitoyl-CoA was ϳ40% lower in lung than liver (Fig.  2a). This represents the combined activities of LCAD, VLCAD, and ACAD9. Surprisingly, the level of free CoA, which is thought to be rate-limiting for mitochondrial FAO (1, 36), was very low in lung compared with liver and brown adipose tissue (Fig. 2b). Yet, despite low levels of CoA, ␤-oxidation of palmitate and oleate was readily detected in lung explants, and the rate of FAO matched or exceeded the rate in liver explants (Fig.  2, c and d). The rate of nonmitochondrial FAO was also significantly higher in lung as determined by measuring residual palmitate oxidation in the presence of etomoxir, an irreversible inhibitor of carnitine palmitoyltransferase-1 (Fig.  2c). Etomoxir-insensitive FAO is usually attributed to peroxisomes (37).
LCAD Ϫ/Ϫ Mouse Lungs Demonstrate Significantly Reduced Long-chain FAO-Western blotting confirmed the absence of LCAD antigen in lung homogenates prepared from LCAD Ϫ/Ϫ mice (Fig. 3a). VLCAD and ACAD9, which also dehydrogenate long-chain acyl-CoAs, were slightly increased in the LCAD Ϫ/Ϫ lung as observed by Western blot, but these changes did not reach statistical significance (Fig. 3b). Total long-chain ACAD activity in lung homogenates from LCAD Ϫ/Ϫ mice was reduced by 50 and 40% with palmitoyl-CoA and oleoyl-CoA as substrates, respectively (Fig. 3c). Accordingly, flux through the FAO pathway as measured with radiolabeled palmitate and oleate was also significantly reduced in lung explants from LCAD Ϫ/Ϫ mice (Fig. 3d). However, oxidation of octanoate (C8), a medium-chain fatty acid that is not a substrate for LCAD, was not reduced in LCAD Ϫ/Ϫ lung explants (Fig. 3e).
LCAD Ϫ/Ϫ Mice Have Altered Lung Mechanics-Lung function in nonstressed LCAD Ϫ/Ϫ mice was determined using a mechanical ventilator (FlexiVent). First, baseline values for the parameters of central airway resistance, tissue resistance, and tissue elastance were measured. Modest but statistically significant changes were seen in baseline lung tissue resistance and elastance in LCAD Ϫ/Ϫ mice, although central airway resistance was not altered (Fig. 4a). Thus, LCAD Ϫ/Ϫ lungs are stiffer and more difficult to inflate, with greater elastic recoil. To determine whether these changes were associated with airway hyper-responsiveness, another cohort of animals was subjected to methacholine-induced bronchoconstriction. LCAD Ϫ/Ϫ and wild-type mice responded similarly to increasing doses of methacholine with regard to central airway resistance and tissue elastance (data not shown). LCAD Ϫ/Ϫ mice were hyporesponsive on the tissue resistance parameter (Fig. 4b), indicating that the change in baseline lung mechanics is not likely due to heightened airway responsiveness. Similarly, H&E-stained Actin and GAPDH were differentially expressed between the different cell types, and thus Ponceau S staining of the blot is shown as a loading control (bottom panel). c, caveolin-1 expression, a marker of AT1 cells, in 5 g of lysate from freshly isolated ATII cells versus the same cells trans-differentiated into ATI-like cells. d, representative Western blot comparing LCAD antigen levels in 5 g of extract prepared from freshly isolated ATII cells (D0) versus the same cells after 6 days in culture (D6) or after trans-differentiation into ATI-like cells. The bar graph represents the means Ϯ S.D. of LCAD normalized to actin in three sets of D0, D6, and ATI-like cells. *, p Ͻ 0.05 versus ATII-D0. FIGURE 2. Lung tissue oxidizes fatty acids at a high rate. All graphs depict means Ϯ S.D. a, acyl-CoA dehydrogenase enzyme activity toward palmitoyl-CoA (combined activities of LCAD, VLCAD, and ACAD9) was determined in lung and liver tissue homogenates. n ϭ 5 mice per group; *, p Ͻ 0.01. b, free co-enzyme A, an essential cofactor for mitochondrial FAO, was measured in mouse tissue extracts using HPLC. Liver and lung both have much lower CoA levels than brown adipose tissue. n ϭ 3 mice, *, p Ͻ 0.01. c, [ 3 H]palmitate (C16:0) oxidation in minced lung versus liver (n ϭ 4 mice). Tissues were treated with etomoxir (Eto) as an inhibitor of mitochondrial FAO or DMSO vehicle (Veh). Etomoxir-insensitive FAO is considered peroxisomal in origin. *, p Ͻ 0.01 for etomoxir versus vehicle; #, p Ͻ 0.01 for lung versus liver. d, [ 3 H]oleate, an unsaturated fatty acid (C18:1), is oxidized at comparable rates between mouse liver and lung explants (n ϭ 4 mice).
sections of lung tissue from LCAD Ϫ/Ϫ mice did not show obvious signs of pathology or the presence of immune cells (Fig. 4c), eliminating chronic inflammation as the cause of the altered lung mechanics.
Pressure-volume curves were analyzed to calculate static compliance and hysteresis. Pulmonary compliance was significantly reduced in LCAD Ϫ/Ϫ mice, again indicating stiff lungs that are more difficult to inflate (Fig. 4d). Consistent with altered breathing mechanics, the area under the pressure-volume curves during inspiration versus expiration (hysteresis) was significantly greater in LCAD Ϫ/Ϫ mice (Fig. 4e).
Lavage Fluid from LCAD Ϫ/Ϫ Mice Shows Increased Protein Content and Decreased Phosphatidylcholine-The epithelial lining fluid at the tissue-air interface in alveoli contains pulmonary surfactant, immune cells, and many proteins either secreted by the lung epithelium or transported across the epithelial barrier from blood (38,39). Epithelial lining fluid was collected from LCAD Ϫ/Ϫ mice by bronchoalveolar lavage with saline. The amount of saline recovered during lavage was recorded and was not significantly different between genotypes. Immune cells were collected from lavage fluid by centrifugation, stained, and counted. As typical for immunologically naive mice, only alveolar macrophages were observed, with significantly fewer recovered from LCAD Ϫ/Ϫ mice (Fig. 5a). The cell-free supernatant from LCAD Ϫ/Ϫ mice contained significantly more total protein (Fig. 5b) and more serum albumin per ml (Fig. 5c) suggesting increased epithelial permeability to serum proteins. Next, the concentration of total PC, the major surfactant phospholipid component, was measured and found to be reduced in lavage fluid from LCAD Ϫ/Ϫ mice (Fig. 5d).
Amount and Function of Pulmonary Surfactant Is Reduced in LCAD Ϫ/Ϫ Mice-To further investigate the possibility of surfactant deficiency in LCAD Ϫ/Ϫ mice, lavage fluids were subjected to centrifugation to isolate the large aggregate, surfaceactive surfactant fraction (40). The amount of phospholipids in the large aggregate fraction was determined using a phosphorus assay and was found to be significantly reduced in LCAD Ϫ/Ϫ mice (Fig. 6a). Next, lipid extracts from large aggregate surfactant were subjected to high performance thin layer chromatography followed by mass spectrometry to quantify the molecular species of PC and PG, which are the major phospholipid classes in surfactant. Several species showed significant differences between genotypes, and these are listed in Table 1. LCAD Ϫ/Ϫ mice displayed reduced dipalmitoyl (C16:0/C16:0)-PC concomitant with increased abundance of PC species with longer and unsaturated acyl chains. Similar changes were seen for PG. To determine whether these changes in composition alter surfactant function, the biophysical properties of the large aggregate surfactant were investigated using a constrained drop surfactometer (28). Surfactant from wild-type and LCAD Ϫ/Ϫ mice was subjected to compression-expansion cycles at a rate of 3 s per cycle, which mimics the rate of human breathing (mice breathe ϳ10ϫ faster). Surface tension was monitored during the dynamic cycling. The maximum and minimum surface tensions achieved during cycling did not differ between genotypes of mice (Fig. 6, b and c). However, LCAD Ϫ/Ϫ surfactant showed higher compressibility, requiring greater compression to reach minimum surface tension (Fig. 6d). Similarly, the surface tension measured at a 20% compression ratio of the surface area, considered to mimic alveolar compression during breathing (41), was significantly higher in LCAD Ϫ/Ϫ mice (Fig. 6e).
LCAD Ϫ/Ϫ Lung Explants Synthesize Phospholipids Normally-Synthesis and trafficking of surfactant is an energy-consuming process (42), and we hypothesized that reduced mitochondrial energy metabolism in LCAD Ϫ/Ϫ lung might limit surfactant synthesis. To test this hypothesis, the incorporation of radiolabeled palmitate and choline into phospholipids was measured in fresh lung explants from wild-type and LCAD Ϫ/Ϫ mice. Using either substrate, equal rates of phospholipid synthesis were observed between genotypes of mice (Fig. 7, a and b). Because lung tissue explants include many other cell types besides ATII, we sought to mimic LCAD deficiency in vitro in the murine ATII cell line MLE12 using the FAO inhibitor etomoxir. Etomoxir inhibited [ 3 H]palmitate oxidation by 75% in FIGURE 4. LCAD ؊/؊ mice have altered lung mechanics. All graphs depict means Ϯ S.D. a, baseline lung mechanics in unstressed wild-type (ϩ/ϩ) versus LCAD knock-out (Ϫ/Ϫ) mice as determined by FlexiVent. A total of 15 animals was measured over two sessions, and the data were combined by normalizing to the mean of the ϩ/ϩ mice in each session. *, p Ͻ 0.05. b, LCAD Ϫ/Ϫ mice are hypo-responsive to methacholine on the FlexiVent tissue resistance (G) parameter. Mice (n ϭ 5) were challenged with increasing doses of methacholine during mechanical ventilation by FlexiVent. The elastance and airway resistance variables were the same between genotypes (data not shown); *, p Ͻ 0.05. c, representative H&E-stained lung sections from an LCAD Ϫ/Ϫ mouse and a wild-type control mouse. LCAD Ϫ/Ϫ lung shows no obvious signs of pathology. d and e, pulmonary compliance (n ϭ 8) and hysteresis (n ϭ 8) were significantly lower and higher, respectively in LCAD Ϫ/Ϫ mice; *, p Ͻ 0.05. FIGURE 5. Lavage fluid from LCAD ؊/؊ mice shows increased protein content and decreased phosphatidylcholine. All bar graphs represent means Ϯ S.D. a, mice (n ϭ 4) were lavaged with three 1-ml aliquots of saline, and immune cells were collected by centrifugation, counted, and stained. Macrophages were the only cell type observed in both genotypes. *, p Ͻ 0.05. b, cell-free supernatant was subjected to assay for protein concentration. *, p Ͻ 0.01. c, equal volumes of cell-free supernatant were electrophoresed and blotted with anti-albumin antibody. Densitometry was used to quantify the levels of albumin that are shown in the bar graph; a representative blot is shown below the bar graph. *, p Ͻ 0.01. d, lipids were extracted from lavage fluids and subjected to TLC, and the phosphatidylcholine spots were scraped from the plate and analyzed by phosphorus assay (n ϭ 4 mice). *, p Ͻ 0.05. All of these measures (a-d) were repeated in a second cohort of mice with similar results.
Increased surfactant degradation could also cause the observed reduction in surfactant phospholipids. Secreted PLA 2 is a known mechanism of surfactant degradation that has been implicated in respiratory diseases (43,44). PLA 2 activity in lavage fluid from LCAD Ϫ/Ϫ and wild-type mice was very low in both genotypes and could not be accurately determined. Likewise, PLA 2 was difficult to detect by Western blotting of lavage FIGURE 6. Amount and function of pulmonary surfactant is reduced in LCAD ؊/؊ mice. a, large aggregate, surface-active pulmonary surfactant was isolated from lavage fluid (n ϭ 4 mice). Total lipids were extracted from the large aggregate material, and phospholipids were quantified by phosphorus assay. Shown are means Ϯ S.D. *, p Ͻ 0.01. b-e, large aggregate material was assayed for surfactant function on a constrained drop surfactometer. Phospholipid concentrations were adjusted to 1.0 mg/ml, and the samples were dynamically cycled at varying compression ratios. Three mice of each genotype were analyzed in triplicate, and the bar graphs represent means Ϯ S.E. b and c, minimum and maximum surface tensions achieved during cycling did not differ between genotypes. d, LCAD Ϫ/Ϫ mouse surfactant showed greater compressibility as demonstrated by requiring a higher compression ratio to reach minimum surface tension. *, p Ͻ 0.01. e, surface tension at a physiological compression ratio (20%) is significantly higher in LCAD Ϫ/Ϫ surfactant. *, p Ͻ 0.01. fluid and was only sporadically observed in fluids from both genotypes (blots not shown). These data pointing to low PLA 2 in both genotypes of mice, although not conclusive, suggest that PLA 2 is not the mechanism behind reduced lung function in LCAD Ϫ/Ϫ mice.

Loss of LCAD Antigen in Two Cases of Sudden Unexplained Infant
Death-We postulate that LCAD may play a role in human lung pathophysiology. Disorders of fatty acid metabolism have previously been linked to sudden infant death syndrome (45). We used Western blotting to evaluate LCAD antigen in samples of lung from six infants who suffered unexplained deaths under the age of 1 year. Two of the six infants had no detectable LCAD antigen (Fig. 8a). The two samples of infant lung with no detectable LCAD were both positive for MCAD, a closely related enzyme to LCAD, as well as surfactant protein-B expression demonstrating the presence of ATII cells in the archived lung materials (Fig. 8a, bottom panel). Next, quantitative PCR was used to measure LCAD mRNA in Cases 1-3 using the antigen-positive Case 2 as the reference for normalization. Compared with Case 2, Case 1 had ϳ40% less LCAD mRNA, whereas Case 3 was similar to Case 2. Genomic DNA was isolated from remaining lung tissue and used for sequencing the 11 LCAD exons and intron-exon boundaries. Cases 1 and 3 were both homozygous for a polymorphism that changes lysine residue 333 to glutamine. Case 2 was heterozygous for this polymorphism. No other mutations were detected.

DISCUSSION
These studies are the first to link FAO disorders to pulmonary lipid metabolism and lung dysfunction. Using human lung tissue and human primary cells, we demonstrated that the orphan FAO enzyme LCAD is enriched in surfactant-producing ATII cells. Subsequent mouse studies confirmed a physiological role for LCAD and the FAO pathway in lung. Lung FAO was found to be robust with rates of palmitate and oleate oxidation that rival that of the liver. LCAD knock-out mice showed reduced pulmonary compliance due to a paucity of large aggregate surfactant. Furthermore, the surfactant that is present in LCAD mice has altered molecular composition and a reduced ability to lower surface tension, and demonstrates greater compressibility.
In humans, surfactant deficiency manifests as respiratory distress syndrome (RDS), which is a major cause of mortality in children less than 1 year old (46). RDS is particularly common among premature infants because surfactant production by ATII cells occurs primarily in the final weeks of gestation. When surfactant deficiency occurs in term infants, the cause is often genetic. To date, LCAD has not been associated with RDS. However, RDS has been noted in several children deficient in mitochondrial trifunctional protein, the enzyme that functions just downstream of LCAD in the FAO pathway (47,48). In one study of mitochondrial trifunctional protein patients, pneumonia and respiratory failure contributed to the deaths of six out of 13 patients followed (49). This link between mitochondrial trifunctional protein and respiratory failure suggests an important role for FAO in the lung. In this study, we evaluated postmortem lung tissue from a small cohort of infants who suffered sudden unexplained deaths. Two of these cases had no detectable LCAD antigen. Sequencing revealed them both to be homozygous for a common single nucleotide polymorphism (rs2286963) resulting in an amino acid substitution (K333Q). In the dbSNP database (www.ncbi.nlm.nih.gov), 30.5% of 1270 individuals genotyped were heterozygous for K333Q, and 5.2% were homozygous. The probability of selecting two tissue samples at random that are homozygous for this polymorphism is ϳ1/400. Although no functional studies have been done for the K333Q variant, recent studies have linked it to increases in blood levels of C9-carnitine (50,51). C9-carnitine is likely to be 2,6-dimethylheptanoyl-carnitine, a branched-chain acylcarnitine that would be predicted to accumulate in LCAD deficiency (5,52). Future studies will determine the impact of the K333Q polymorphism on enzyme activity and stability, as well as establish the frequency of the polymorphism among cases of RDS and unexplained sudden infant deaths. We postulate that homozygosity for K333Q may increase the risk for infant mortality from pulmonary infections or other forms of alveolar stress.
Environmental or dietary factors may also suppress the FAO pathway in lung. For instance, conditions that deplete carnitine can severely limit FAO, which is carnitine-dependent. Lung cannot synthesize carnitine and must therefore transport it via FIGURE 8. Loss of LCAD antigen in two cases of sudden unexplained infant death. a, anti-LCAD Western blotting of 50 g of total protein extract prepared from post-mortem lung tissues from six children who suffered sudden deaths in the 1st year of life. The extracts from Cases 1 to 3 were further evaluated for MCAD and surfactant protein-B (SP-B) precursor expression. b, quantitative PCR was used to compare LCAD mRNA levels between Cases 1-3, normalized to Case 2 as a normal control. Sequencing of genomic DNA revealed the presence of the K333Q polymorphism; the genotype of each case with regard to this polymorphism is indicated on the bars with white font. c, Lys-333 is just downstream of two lysines (Lys-318/Lys-322) known to be regulated by reversible acetylation (69) and is highly conserved across species.
the transporter protein OCTN2 that is expressed in the alveolar epithelium (53). Besides carnitine, OCTN2 transports many cationic drugs, including ipratropium, which is an inhaled therapeutic for COPD and asthma (54). Competitive inhibition of OCTN2 by drugs can result in secondary carnitine deficiency (54). OCTN2 has been shown to be competitively inhibited to various degrees by dozens of drugs, including anti-cancer agents, ␤-lactam antibiotics, statins, ␤-blockers, antidepressants, and antihistamines, among others (55)(56)(57)(58). The effects of these drugs on carnitine transport and FAO in the lung epithelium in vivo have not been established and require further investigation, especially in light of recent studies demonstrating carnitine deficiency in both children and adults with pulmonary disease (59 -62).
The dominant phospholipid species in surfactant is dipalmitoyl-PC, which contains two 16-carbon saturated acyl chains (63). We have shown that disruption of LCAD in mice leads to changes in PC and PG composition as observed by mass spectrometry (Table 1), and these changes are associated with changes in surfactant behavior in the constrained drop surfactometer. To our knowledge, this is the first analysis of the phospholipid molecular species in the LCAD Ϫ/Ϫ mouse model. Previous studies have measured free fatty acids in the liver and serum acylcarnitines and noted an accumulation of 14-carbon lipids, particularly free C14:1 fatty acids and C14:1-carnitine (64). In contrast, the changes in surfactant composition reported here involve an increase in C18 to C22 chain lengths with varying degrees of unsaturation, and C14:1 was not detected in either PC or PG. Clearly, if C14:1 accumulates in lung of LCAD Ϫ/Ϫ mice, it is not incorporated into surfactant. Further work will be necessary to determine whether accumulating C14:1 may be elongated and further desaturated to produce the C18:1, C18:2, and C20:4 acyl chains observed in LCAD Ϫ/Ϫ surfactant or whether LCAD and the mitochondrial FAO pathway are important for chain-shortening C18 -C22 fatty acids down to C16 acyl chains for incorporation into surfactant. Interestingly, mice deficient in the elongation of longchain fatty acid family member 6 (Elovl6), an enzyme that elongates unsaturated fatty acids, are prone to lung fibrosis (65) suggesting that the proper mixture of phospholipid species is essential for pulmonary health.
Although the qualitative changes seen in LCAD Ϫ/Ϫ surfactant during dynamic cycling are most likely linked to the changes in phospholipid acyl chain composition, a clear mechanism is lacking for the quantitative change in surfactant phospholipids. The depletion in large aggregate surfactant phospholipids is probably the primary factor behind the reduced pulmonary compliance observed in LCAD Ϫ/Ϫ animals. This depletion is likely due to decreased surfactant synthesis or increased recycling/degradation. Our lung explant studies showed normal rates of choline incorporation into total lipid, but we did not investigate trafficking of lamellar bodies and extracellular release of surfactant material. With regard to degradation, we found no observable increase in PLA 2 , which actively degrades phospholipids and is known to be increased in lavage fluid of children with RDS (66). Albumin, however, was consistently increased in LCAD Ϫ/Ϫ lavage fluid, and albumin has been shown to inactivate surfactant and to possess a phos-pholipase-like activity (67,68). Loss of FAO as an energy source in ATI and ATII cells may compromise the integrity of the alveolar epithelium, leading to increased albumin and other proteins in the epithelial lining fluid, which may then inactivate or disrupt pulmonary surfactant. Further studies are underway to investigate these aspects of surfactant metabolism in LCAD Ϫ/Ϫ mice and to evaluate LCAD as a candidate gene for pulmonary disease in humans.