Multi-omic analysis of the effects of low frequency ventilation during cardiopulmonary bypass surgery.

BACKGROUND: Heart surgery with cardio-pulmonary bypass (CPB) is associated with lung ischemia leading to injury and inflammation. It has been suggested this is a result of the lungs being kept deflated throughout the duration of CPB. Low frequency ventilation (LFV) during CPB has been proposed to reduce lung dysfunction. METHODS: We used a semi-biased multi-omic approach to analyse lung biopsies taken before and after CPB from 37 patients undergoing coronary artery bypass surgery randomised to both lungs left collapsed or using LFV for the duration of CPB. We also examined inflammatory and oxidative stress markers from blood samples from the same patients. RESULTS: 30 genes were induced when the lungs were left collapsed and 80 by LFV. Post-surgery 26 genes were significantly higher in the LFV vs. lungs left collapsed, including genes associated with inflammation (e.g. IL6 and IL8) and hypoxia/ischemia (e.g. HIF1A, IER3 and FOS). Relatively few changes in protein levels were detected, perhaps reflecting the early time point or the importance of post-translational modifications. However, pathway analysis of proteomic data indicated that LFV was associated with increased "cellular component morphogenesis" and a decrease in "blood circulation". Lipidomic analysis did not identify any lipids significantly altered by either intervention. DISCUSSION: Taken together these data indicate the keeping both lungs collapsed during CPB significantly induces lung damage, oxidative stress and inflammation. LFV during CPB increases these deleterious effects, potentially through prolonged surgery time, further decreasing blood flow to the lungs and enhancing hypoxia/ischemia.

A robust and versatile mass spectrometry platform for comprehensive assessment of the thiol redox metabolome.

Several diseases are associated with perturbations in redox signaling and aberrant hydrogen sulfide metabolism, and numerous analytical methods exist for the measurement of the sulfur-containing species affected. However, uncertainty remains about their concentrations and speciation in cells/biofluids, perhaps in part due to differences in sample processing and detection principles. Using ultrahigh-performance liquid chromatography in combination with electrospray-ionization tandem mass spectrometry we here outline a specific and sensitive platform for the simultaneous measurement of 12 analytes, including total and free thiols, their disulfides and sulfide in complex biological matrices such as blood, saliva and urine. Total assay run time is < 10 min, enabling high-throughput analysis. Enhanced sensitivity and avoidance of artifactual thiol oxidation is achieved by taking advantage of the rapid reaction of sulfhydryl groups with N-ethylmaleimide. We optimized the analytical procedure for detection and separation conditions, linearity and precision including three stable isotope labelled standards. Its versatility for future more comprehensive coverage of the thiol redox metabolome was demonstrated by implementing additional analytes such as methanethiol, N-acetylcysteine, and coenzyme A. Apparent plasma sulfide concentrations were found to vary substantially with sample pretreatment and nature of the alkylating agent. In addition to protein binding in the form of mixed disulfides (S-thiolation) a significant fraction of aminothiols and sulfide appears to be also non-covalently associated with proteins. Methodological accuracy was tested by comparing the plasma redox status of 10 healthy human volunteers to a well-established protocol optimized for reduced/oxidized glutathione. In a proof-of-principle study a deeper analysis of the thiol redox metabolome including free reduced/oxidized as well as bound thiols and sulfide was performed. Additional determination of acid-labile sulfide/thiols was demonstrated in human blood cells, urine and saliva. Using this simplified mass spectrometry-based workflow the thiol redox metabolome can be determined in samples from clinical and translational studies, providing a novel prognostic/diagnostic platform for patient stratification, drug monitoring, and identification of new therapeutic approaches in redox diseases.

Acute respiratory distress syndrome and acute lung injury.

Acute respiratory distress syndrome (ARDS) is a life threatening respiratory failure due to lung injury from a variety of precipitants. Pathologically ARDS is characterised by diffuse alveolar damage, alveolar capillary leakage, and protein rich pulmonary oedema leading to the clinical manifestation of poor lung compliance, severe hypoxaemia, and bilateral infiltrates on chest radiograph. Several aetiological factors associated with the development of ARDS are identified with sepsis, pneumonia, and trauma with multiple transfusions accounting for most cases. Despite the absence of a robust diagnostic definition, extensive epidemiological investigations suggest ARDS remains a significant health burden with substantial morbidity and mortality. Improvements in outcome following ARDS over the past decade are in part due to improved strategies of mechanical ventilation and advanced support of other failing organs. Optimal treatment involves judicious fluid management, protective lung ventilation with low tidal volumes and moderate positive end expiratory pressure, multi-organ support, and treatment where possible of the underlying cause. Moreover, advances in general supportive measures such as appropriate antimicrobial therapy, early enteral nutrition, prophylaxis against venous thromboembolism and gastrointestinal ulceration are likely contributory reasons for the improved outcomes. Although therapies such as corticosteroids, nitric oxide, prostacyclins, exogenous surfactants, ketoconazole and antioxidants have shown promising clinical effects in animal models, these have failed to translate positively in human studies. Most recently, clinical trials with ß2 agonists aiding alveolar fluid clearance and immunonutrition with omega-3 fatty acids have also provided disappointing results. Despite these negative studies, mortality seems to be in decline due to advances in overall patient care. Future directions of research are likely to concentrate on identifying potential biomarkers or genetic markers to facilitate diagnosis, with phenotyping of patients to predict outcome and treatment response. Pharmacotherapies remain experimental and recent advances in the modulation of inflammation and novel cellular based therapies, such as mesenchymal stem cells, may reduce lung injury and facilitate repair.

Cortisol clearance and associations with insulin sensitivity, body fat and fatty liver in middle-aged men.

AIMS/HYPOTHESIS: The regulation of cortisol metabolism in vivo is not well understood. We evaluated the relationship between cortisol metabolism and insulin sensitivity, adjusting for total and regional fat content and for non-alcoholic fatty liver disease. MATERIALS AND METHODS: Twenty-nine middle-aged healthy men with a wide range of BMI were recruited. We measured fat content by dual-energy X-ray absorptiometry and magnetic resonance imaging (MRI), liver fat by ultrasound and MRI, the hypothalamic-pituitary-adrenal axis by adrenal response to ACTH(1-24), unconjugated urinary cortisol excretion, corticosteroid-binding globulin, and cortisol clearance by MS. We assessed insulin sensitivity by hyperinsulinaemic-euglycaemic clamp and by OGTT. RESULTS: Cortisol clearance was strongly inversely correlated with insulin sensitivity (M value) (r = -0.61, p = 0.002). Cortisol clearance was increased in people with fatty liver compared with those without (mean+/-SD: 243 +/- 10 vs 158 +/- 36 ml/min; p = 0.014). Multiple regression modelling showed that the relationship between cortisol clearance and insulin sensitivity was independent of body fat. The relationship between fatty liver and insulin sensitivity was significantly influenced by body fat and cortisol clearance. CONCLUSIONS/INTERPRETATION: Cortisol clearance is strongly associated with insulin sensitivity, independently of the amount of body fat. The relationship between fatty liver and insulin sensitivity is mediated in part by both fatness and cortisol clearance.

Mass spectroscopic analysis of phosphatidylinositol synthesis using 6-deuteriated-myo-inositol: comparison of the molecular specificities and acyl remodelling mechanisms in mouse tissues and cultured cells.

Mammalian cell PtdIns (phosphatidylinositol) in vivo is enriched in the sn-1-stearoyl 2-arachidonoyl species, the physiological precursor of phosphatidylinositol 4,5-bisphosphate. Mechanisms regulating this specificity are unclear but are typically lost for cells in culture. We used ESI-MS (tandem electrospray ionization-mass spectrometry) to determine the molecular species of PtdIns synthesized by mouse tissues in vivo compared with cultured cells in vitro. After incorporation of deuteriated myo-d(6)-inositol over 3 h, endogenous and newly synthesized PtdIns and lysoPtdIns species were quantified from precursor scans of m/z 241(-) and m/z 247(-) respectively. PtdIns was synthesized as a wide range of species irrespective of the final membrane composition. Analyses of isotope enrichments argued against acyl remodelling as the major regulatory mechanism: composition of the lysoPtdIns pool under all conditions reflected that of either endogenous or newly synthesized PtdIns and was always at equilibrium. The kinetics of PtdIns synthesis, together with the prolonged time scale required for achieving final equilibrium compositions suggest that selective transport between membranes and/or hydrolysis of selected molecular species are the most probable mechanisms regulating compositions of PtdIns and, ultimately, phosphatidylinositol 4,5-bisphosphate.

Lipidomic analysis of the molecular specificity of a cholinephosphotransferase in situ.

Dynamic lipidomics using ESI-MS (tandem electrospray ionization mass spectrometry) of 9-deuterated choline (choline-d(9)) incorporation into mammalian cell PtdCho (phosphatidylcholine) permits assessment of the molecular specificity of synthesis. Bulk cell PtdCho synthesis occurs in spatially distinct locations, using separate CPTs (1,2 diacylglycerol CDP:choline cholinephosphotransferases). We assessed whether in vitro molecular selectivity of DAG (diacylglycerol) incorporation between CPTs is manifest in situ, by monitoring choline-d(9) incorporation into PtdCho and lyso-PtdCho molecular species over 3 h in control cells and in CHO-K1 cells overexpressing hCEPT1. Compared with controls, the basal molecular species composition of hCEPT1 overexpressors was significantly enriched in arachidonate. This was not due to net accretion of cellular PtdCho arguing against effects of inadequate unsaturated PtdCho degradation or remodelling. Rather, time-course analyses of PtdCho and lyso-PtdCho pools showed that both arachidonate-containing DAG incorporation and turnover of PtdCho is increased in hCEPT1 overexpressors. Increased choline-d(9) incorporation into arachidonyl lyso-PtdCho shows that both phospholipase A(1)- and A(2)-mediated turnover is involved. Spatially distinct molecular specificity of DAG incorporation into cellular PtdCho at the level of hCEPT1 exists in situ.

Use of mass spectrometry-based lipidomics to probe PITPalpha (phosphatidylinositol transfer protein alpha) function inside the nuclei of PITPalpha+/+ and PITPalpha-/- cells.

The mammalian phospholipid exchange protein PITPalpha (phosphatidylinositol transfer protein alpha), found in both extranuclear and endonuclear compartments, is thought in part to facilitate nuclear import of the PtdIns (phosphatidylinositol) consumed in the generation of proliferation-associated endonuclear diacylglycerol accumulations. Unlike phosphatidylcholine, endonuclear PtdIns is not synthesized in situ. However, despite progressive postnatal lethality of PITPalpha ablation in mice, PITPalpha(-/-) MEF (mouse embryonic fibroblasts) lack an obviously impaired proliferative capacity. We used ESI-MS (tandem electrospray ionization-MS) to monitor incorporation of the deuterated phospholipid precursors, choline-d(9) and inositol-d(6), into molecular species of whole cell and endonuclear phosphatidylcholine and PtdIns over 24 h to assess the contribution of PITPalpha to the nuclear import of PtdIns into MEF cells. In cells labelled for 1, 3, 6, 12 and 24 h fractional inositol-d(6) incorporation into whole-cell PtdIns species was consistently higher in PITPalpha(-/-) MEF implying greater flux through its biosynthetic pathway. Moreover, endonuclear accumulation of PtdIns-d(6) was apparent in the PITPalpha(-/-) cells and mirrored that in PITPalpha(+/+) cells. Together, these results suggest that the essential endonuclear PtdIns import via PITPalpha can be accommodated by other mechanisms.

The effect of eicosapentaenoic acid on rat lymphocyte proliferation depends upon its position in dietary triacylglycerols.

Animal and human studies have shown that greatly increasing the amount of fish oil [rich in long-chain (n-3) PUFA] in the diet can decrease lymphocyte functions. The effects of a more modest provision of long-chain (n-3) PUFA and whether eicosapentaenoic acid (20:5) and docosahexaenoic acid (22:6) have the same effects as one another are unclear. Whether the position of 20:5 or 22:6 in dietary triacylglycerols (TAG) influences their incorporation into immune cells and their subsequent functional effects is not known. In this study, male weanling rats were fed for 6 wk one of 9 diets that contained 178 g lipid/kg and that differed in the type of (n-3) PUFA and in the position of these in dietary TAG. The control diet contained 4.4 g alpha-linolenic acid (18:3)/100 g total fatty acids. In the other diets, 20:5 or 22:6 replaced a portion (50 or 100%) of 18:3, and were in the sn-2 or the sn-1(3) position of dietary TAG. There were significant dose-dependent increases in the proportion of 20:5 or 22:6 in spleen mononuclear cell phospholipids when 20:5 or 22:6 was fed. These increases were at the expense of arachidonic acid and were largely independent of the position of 20:5 or 22:6 in dietary TAG. Spleen lymphocyte proliferation increased dose dependently when 20:5 was fed in the sn-1(3) position of dietary TAG. There were no significant differences in interleukin-2, interferon-gamma or interleukin-10 production among spleen cells from rats fed the different diets. Prostaglandin E(2) production by spleen mononuclear cells was decreased by inclusion of either 20:5 or 22:6 in the diet in the sn-1(3) position. Thus, incorporation of 20:5 or 22:6 into spleen mononuclear cell phospholipids is not influenced by the position in dietary TAG. However, the pattern of incorporation may be influenced, and there are some differential functional effects of the position of long-chain (n-3) PUFA in dietary TAG. A moderate increase in the intake of 20:5 at the sn-1(3) position of dietary TAG increases lymphocyte proliferation.

Electrospray ionisation mass spectrometric analysis of lipid restructuring in the carp (Cyprinus carpio L.) during cold acclimation.

Cold acclimation of carp from 30 degrees C to 10 degrees C causes a restructuring of liver microsomal phospholipids characterised by increased proportions of monounsaturated fatty acid in phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Here, we have used electrospray ionisation mass spectrometry (ESI-MS) to determine the patterns of alteration to individual molecular species compositions of PC, PE and phosphatidylinositol (PI) in response to gradually decreasing temperature. The results demonstrate that cold induces precise changes to a limited number of phospholipid species, and that these changes are distinct and different for each phospholipid class. The major change for PC was increased 16:1/22:6, but for PE the species that increased was 18:1/22:6. By contrast, the PI species that increased during cold acclimation were characterised by an sn-1 monounsaturated fatty acid in combination with arachidonoyl or eicosapentaenoyl fatty acid at the sn-2 position. Analysis of acyl distribution indicates that cold only caused the accumulation of monounsaturated fatty acids at the sn-1 and not at the sn-2 position of phospholipids. These results highlight the tight and restricted range of modifications that membranes make to their phospholipid composition in response to thermal stress.

Pulmonary surfactant in birds: coping with surface tension in a tubular lung.

As birds have tubular lungs that do not contain alveoli, avian surfactant predominantly functions to maintain airflow in tubes rather than to prevent alveolar collapse. Consequently, we have evaluated structural, biochemical, and functional parameters of avian surfactant as a model for airway surfactant in the mammalian lung. Surfactant was isolated from duck, chicken, and pig lung lavage fluid by differential centrifugation. Electron microscopy revealed a uniform surfactant layer within the air capillaries of the bird lungs, and there was no tubular myelin in purified avian surfactants. Phosphatidylcholine molecular species of the various surfactants were measured by HPLC. Compared with pig surfactant, both bird surfactants were enriched in dipalmitoylphosphatidylcholine, the principle surface tension-lowering agent in surfactant, and depleted in palmitoylmyristoylphosphatidylcholine, the other disaturated phosphatidylcholine of mammalian surfactant. Surfactant protein (SP)-A was determined by immunoblot analysis, and SP-B and SP-C were determined by gel-filtration HPLC. Neither SP-A nor SP-C was detectable in either bird surfactant, but both preparations of surfactant contained SP-B. Surface tension function was determined using both the pulsating bubble surfactometer (PBS) and capillary surfactometer (CS). Under dynamic cycling conditions, where pig surfactant readily reached minimal surface tension values below 5 mN/m, neither avian surfactant reached values below 15 mN/m within 10 pulsations. However, maximal surface tension of avian surfactant was lower than that of porcine surfactant, and all surfactants were equally efficient in the CS. We conclude that a surfactant composed primarily of dipalmitoylphosphatidylcholine and SP-B is adequate to maintain patency of the air capillaries of the bird lung.

A comparison of the molecular species compositions of mammalian lung surfactant phospholipids.

While dipalmitoyl phosphatidylcholine (PC16:0/16:0) is essential for pulmonary surfactant function, roles for other individual molecular species of surfactant phospholipids have not been established. If any phospholipid species other than PC16:0/16:0 is important for surfactant function, then it may be conserved across animal species. Consequently, we have quantified, by electrospray ionisation mass spectrometry, molecular species compositions of phosphatidylcholine (PC), phosphatidylglycerol (PG) and phosphatidylinositol (PI) in surfactants from human, rabbit, rat and guinea pig lungs. While PC compositions displayed only relatively minor variations across the animal species studied, there were wide variations of PG and PI concentrations and compositions. Human surfactant PG and PI were enriched in the same three monounsaturated species (PG16:0/18:1, PG18:1/18:1 and PG18:0/18:1) with minimal amounts of PG16:0/16:0 or polyunsaturated species, while all animal surfactant PG contained increased concentrations of PG16:0/16:0 and PG16:0/18:2. Animal surfactant PIs were essentially monounsaturated except for a high content of PI18:0/20:4 (29%) in the rat. As these four surfactants all maintain appropriate lung function of the respective animal species, then all their varied compositions of acidic phospholipids must be adequate at promoting the processes of adsorption, film refinement, respreading and collapse characteristic of surfactant. We conclude that this effectively monounsaturated composition of anionic phospholipid molecular species is a common characteristic of mammalian surfactants.

Pulmonary and gastric surfactants. A comparison of the effect of surface requirements on function and phospholipid composition.

Surfactant is present in the alveoli and conductive airways of mammalian lungs. The presence of surface active agents was, moreover, demonstrated for avian tubular lungs and for the stomach and intestine. As the surface characteristics of these organs differ from each other, their surfactants possess distinct biochemical and functional characteristics. In the stomach so-called 'gastric surfactant' forms a hydrophobic barrier to protect the mucosa against acid back-diffusion. For this purpose gastric mucosal cells secrete unsaturated phosphatidylcholines (PC), but no dipalmitoyl-PC (PC16:0/16:0). By contrast, surfactant from conductive airways, lung alveoli and tubular avian lungs contain PC16:0/16:0 as their main component in similar concentrations. Hence, there is no biochemical relation between gastric and pulmonary surfactant. Alveolar surfactant, being designed for preventing alveolar collapse under the highly dynamic conditions of an oscillating alveolus, easily reaches values of <5 mN/m upon cyclic compression. Surfactants from tubular air-exposed structures, however, like the conductive airways of mammalian lungs and the exclusively tubular avian lung, display inferior compressibility as they only reach minimal surface tension values of approximately 20 mN/m. Hence, the highly dynamic properties of alveolar surfactant do not apply for surfactants designed for air-liquid interfaces of tubular lung structures.

Highly saturated endonuclear phosphatidylcholine is synthesized in situ and colocated with CDP-choline pathway enzymes.

Chromatin-associated phospholipids are well recognized. A report that catalytically active endonuclear CTP:choline-phosphate cytidylyltransferase alpha is necessary for cell survival questions whether endonuclear, CDP-choline pathway phosphatidylcholine synthesis may occur in situ. We report that chromatin from human IMR-32 neuroblastoma cells possesses such a biosynthetic pathway. First, membrane-free nuclei retain all three CDP-choline pathway enzymes in proportions comparable with the content of chromatin-associated phosphatidylcholine. Second, following supplementation of cells with deuterated choline and using electrospray ionization mass spectrometry, both the time course and molecular species labeling pattern of newly synthesized endonuclear and whole cell phosphatidylcholine revealed the operation of spatially separate, compositionally distinct biosynthetic routes. Specifically, endogenous and newly synthesized endonuclear phosphatidylcholine species are both characterized by a high degree of diacyl/alkylacyl chain saturation. This unusual species content and synthetic pattern (evident within 10 min of supplementation) are maintained through cell growth arrest by serum depletion and when proliferation is restored, suggesting that endonuclear disaturated phosphatidylcholine enrichment is essential and closely regulated. We propose that endonuclear phosphatidylcholine synthesis may regulate periodic nuclear accumulations of phosphatidylcholine-derived lipid second messengers. Furthermore, our estimates of saturated phosphatidylcholine nuclear volume occupancy of around 10% may imply a significant additional role in regulating chromatin structure.

Exogenous surfactant supplementation in infants with respiratory syncytial virus bronchiolitis.

Infants with respiratory syncytial virus (RSV) bronchiolitis are deficient in surfactant, both in quantity and ability to reduce surface tension. New evidence suggests surfactant has a role in maintaining the patency of conducting airways, which has implications for RSV bronchiolitis. A randomized, controlled pilot study was undertaken to assess the effects of exogenous surfactant supplementation to RSV-positive infants on pulmonary mechanics, indices of gas exchange, and the phospholipid composition of bronchoalveolar lavage fluid (BALF). Nineteen ventilated infants (median corrected age 4 wk) received either two doses of surfactant (Survanta, 100 mg/kg) within 24 and 48 h of mechanical ventilation (n = 9), or air placebo (n = 10). Static lung compliance and resistance of infants in the placebo but not in the surfactant-treated group became progressively worse over the first 30 h following enrollment. Although no significant acute changes in gas exchange parameters were seen following surfactant, infants in the surfactant group showed a more rapid improvement in oxygenation and ventilation indices over the first 60 h of ventilation. Surfactant status was assessed from the concentration ratio in BALF of the disaturated phospholipid species dipalmitoylphosphatidylcholine to that of the monounsaturated species palmitoyloleoylphosphatidylcholine. This ratio correlated with both lung compliance (positively) and resistance (negatively), and over time increased in the treated group and declined in placebo infants. The data from this pilot study suggest that functional surfactant has a role in maintaining small airway patency as well as lung compliance in infants infected with RSV and an outcome study is now warranted.

Altered airway surfactant phospholipid composition and reduced lung function in asthma.

Pulmonary surfactant in bronchoalveolar lavage fluid (BALF) and induced sputum from adults with stable asthma (n = 36) and healthy controls (n = 12) was analyzed for phospholipid and protein compositions and function. Asthmatic subjects were graded as mild, moderate, or severe. Phospholipid compositions of BALF and sputum from control subjects were similar and characteristic of surfactant. For asthmatic subjects, the proportion of dipalmitoyl phosphatidylcholine (16:0/16:0PC), the major phospholipid in surfactant, decreased in sputum (P < 0.05) but not in BALF. In BALF, mole percent 16:0/16:0PC correlated with surfactant function measured in a capillary surfactometer, and sputum mole percent 16:0/16:0PC correlated with lung function (forced expiratory volume in 1 s). Neither surfactant protein A nor total protein concentration in either BALF or sputum was altered in asthma. These results suggest altered phospholipid composition and function of airway (sputum) but not alveolar (BALF) surfactant in stable asthma. Such underlying surfactant dysfunction may predispose asthmatic subjects to further surfactant inhibition by proteins or aeroallergens in acute asthma episodes and contribute to airway closure in asthma. Consequently, administration of an appropriate therapeutic surfactant could provide clinical benefit in asthma.

Phospholipid molecular species of bronchoalveolar lavage fluid after local allergen challenge in asthma.

Electrospray ionization mass spectrometry was used to quantify phosphatidylcholine (PC) and phosphatidylglycerol (PG) molecular species in bronchoalveolar lavage fluid (BALF) from control and mild asthmatic subjects after local allergen challenge. BALF was obtained from 5 control and 13 asthmatic subjects before and 24 h after segmental allergen and saline challenge. There were no differences in the ratio of total PC to total PG or in the molecular species composition of PC or PG between the asthmatic and control groups under basal conditions. Allergen challenge in asthmatic but not in control volunteers caused a significant increase in the PC-to-PG ratio because of increased concentrations of PC species containing linoleic acid (16:0/18:2 PC, 18:0/18:2 PC, and 18:1/18:2 PC). These molecular species were characteristic of plasma PC analyzed from the same subjects, strongly suggesting that the altered PC composition in BALF in asthmatic subjects after allergen challenge was due to infiltration of plasma lipoprotein, not to catabolism of surfactant phospholipid. Interactions between surfactant and lipoprotein infiltrate may contribute to surfactant dysfunction and potentiate disease severity in asthma.

Deficient hydrophilic lung surfactant proteins A and D with normal surfactant phospholipid molecular species in cystic fibrosis.

Chronic bacterial colonization of the lungs, with an excessive inflammatory response, is the major cause of morbidity and mortality in cystic fibrosis. Lung surfactant exhibits a spectrum of potential immunomodulatory properties: phospholipid components inhibit cellular inflammatory responses, whereas the hydrophilic surfactant proteins A (SP-A) and D (SP-D) are integral components of the innate host defense response of the lungs against bacterial infection. Consequently, alteration to the relative proportions of lung surfactant components may alter the susceptibility of the lungs to bacterial colonization. In this study, bronchoalveolar lavage (BAL) samples were collected at diagnostic fiberoptic bronchoscopy from 11 control children, 13 children with cystic fibrosis, and 11 children with acute lung infection. Electrospray ionization mass spectrometry analysis demonstrated negligible changes to the molecular species or total BAL concentrations of phosphatidylcholine, phosphatidylglycerol, or phosphatidylinositol among the three subject groups. In contrast, median SP-A concentration was decreased (P < 0.001) in the cystic fibrosis group (2.65 microg/ml) compared with control (12.35 microg/ml) and infection (9.76 microg/ml) groups. Median SP-D was also decreased (P < 0.05) in the infection (12.17 ng/ml) compared with the control group (641 ng/ml), and was below assay limits for the majority of cystic fibrosis children (P < 0. 001). This dramatic decrease of hydrophilic surfactant proteins in the presence of normal surfactant phospholipid may be one mechanism underlying the relative ineffectiveness of the cellular inflammatory response in killing invading bacteria in the lungs of patients with cystic fibrosis.