Mass spectrometry imaging as a tool for evaluating the pulmonary distribution of exogenous surfactant in premature lambs.

BACKGROUND: The amount of surfactant deposited in the lungs and its overall pulmonary distribution determine the therapeutic outcome of surfactant replacement therapy. Most of the currently available methods to determine the intrapulmonary distribution of surfactant are time-consuming and require surfactant labelling. Our aim was to assess the potential of Mass Spectrometry Imaging (MSI) as a label-free technique to qualitatively and quantitatively evaluate the distribution of surfactant to the premature lamb. METHODS: Twelve preterm lambs (gestational age 126-127d, term ~150d) were allocated in two experimental groups. Seven lambs were treated with an intratracheal bolus of the synthetic surfactant CHF5633 (200 mg/kg) and 5 lambs were managed with mechanical ventilation for 120 min, as controls. The right lung lobes of all lambs were gradually frozen while inflated to 20 cmH2O pressure for lung cryo-sections for MSI analysis. The intensity signals of SP-C analog and SP-B analog, the two synthetic peptides contained in the CHF5633 surfactant, were used to locate, map and quantify the intrapulmonary exogenous surfactant. RESULTS: Surfactant treatment was associated with a significant improvement of the mean arterial oxygenation and lung compliance (p < 0.05). Nevertheless, the physiological response to surfactant treatment was not uniform across all animals. SP-C analog and SP-B analog were successfully imaged and quantified by means of MSI in the peripheral lungs of all surfactant-treated animals. The intensity of the signal was remarkably low in untreated lambs, corresponding to background noise. The signal intensity of SP-B analog in each surfactant-treated animal, which represents the surfactant distributed to the peripheral right lung, correlated well with the physiologic response as assessed by the area under the curves of the individual arterial partial oxygen pressure and dynamic lung compliance curves of the lambs. CONCLUSIONS: Applying MSI, we were able to detect, locate and quantify the amount of exogenous surfactant distributed to the lower right lung of surfactant-treated lambs. The distribution pattern of SP-B analog correlated well with the pulmonary physiological outcomes of the animals. MSI is a valuable label-free technique which is able to simultaneously evaluate qualitative and quantitative drug distribution in the lung.

Immunolocalization of Surfactant Proteins SP-A, SP-B, SP-C, and SP-D in Infantile Labial Glands and Mucosa.

Surfactant proteins in different glandular structures of the oral cavity display antimicrobial activity for protection of invading microorganisms. Moreover, they are involved in lowering liquid tension in fluids and facilitate secretion flows. Numerous investigations for studying the occurrence of surfactant proteins in glandular tissues were performed using different methods. In the oral cavity, minor salivary glands secrete saliva continuously for the maintenance of a healthy oral environment. For the first time, we could show that infantile labial glands show expression of the surfactant proteins (SP) SP-A, SP-B, SP-C, and SP-D in acinar cells and the duct system in different intensities. The stratified squamous epithelium of the oral mucosa revealed positive staining for SPs in various cell layers.

Surfactant proteins in pediatric interstitial lung disease.

BACKGROUND: Children's interstitial lung diseases (chILD) comprise a broad spectrum of diseases. Besides the genetically defined surfactant dysfunction disorders, most entities pathologically involve the alveolar surfactant region, possibly affecting the surfactant proteins SP-B and SP-C. Therefore, our objective was to determine the value of quantitation of SP-B and SP-C levels in bronchoalveolar lavage fluid (BALF) for the diagnosis of chILD. METHODS: Levels of SP-B and SP-C in BALF from 302 children with chILD and in controls were quantified using western blotting. In a subset, single-nucleotide polymorphisms (SNPs) in the SFTPC promoter were genotyped by direct sequencing. RESULTS: While a lack of dimeric SP-B was found only in the sole subject with hereditary SP-B deficiency, low or absent SP-C was observed not only in surfactant dysfunction disorders but also in patients with other diffuse parenchymal lung diseases pathogenetically related to the alveolar surfactant region. Genetic analysis of the SFTPC promoter showed association of a single SNP with SP-C level. CONCLUSION: SP-B levels may be used for screening for SP-B deficiency, while low SP-C levels may point out diseases caused by mutations in TTF1, SFTPC, ABCA3, and likely in other genes involved in surfactant metabolism that remain to be identified. We conclude that measurement of levels of SP-B and SP-C was useful for the differential diagnosis of chILD, and for the precise molecular diagnosis, sequencing of the genes is necessary.

Establishment of LC-MS methods for the analysis of palmitoylated surfactant proteins.

The surfactant proteins (SPs), SP-B and SP-C, are important components of pulmonary surfactant involved in the reduction of alveolar surface tension. Quantification of SP-B and SP-C in surfactant drugs is informative for their quality control and the evaluation of their biological activity. Western blot analysis enabled the quantification of SP-B, but not SP-C, in surfactant drugs. Here, we report a new procedure involving chemical treatments and LC-MS to analyze SP-C peptides. The procedure enabled qualitative analysis of SP-C from different species with discrimination of the palmitoylation status and the artificial modifications that occur during handling and/or storage. In addition, the method can be used to estimate the total amount of SP-C in pulmonary surfactant drugs. The strategy described here might serve as a prototype to establish analytical methods for peptides that are extremely hydrophobic and behave like lipids. The new method provides an easy measurement of SP-C from various biological samples, which will help the characterization of various experimental animal models and the quality control of surfactant drugs, as well as diagnostics of human samples.

Hereditary interstitial lung diseases manifesting in early childhood in Japan.

BACKGROUND: Genetic variations associated with interstitial lung diseases (ILD) have not been extensively studied in Japanese infants. METHODS: Forty-three infants with unexplained lung dysfunction were studied. All 43, 22, and 17 infants underwent analyses of surfactant protein (SP)-C gene (SFTPC) and ATP-binding cassette A3 gene (ABCA3), SP-B gene (SFTPB), and SP-B western blotting, respectively. Two and four underwent assessment of granulocyte macrophage colony-stimulating factor-stimulating phosphorylation of signal transducer and activator of transcription-5 (pSTAT-5) and analyses of FOXF1 gene (FOXF1), respectively. RESULTS: ILD were diagnosed clinically in nine infants: four, three, and two had interstitial pneumonitis, hereditary pulmonary alveolar proteinosis (hPAP), and alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV), respectively. Genetic variations considered responsible were detected in six (67%) of the nine infants with ILD: three with hPAP (SFTPC p.Leu45Arg and p.Gln145fs, and ABCA3 p.Arg1583Trp/p.Val1495CysfsX21), two with interstitial pneumonitis (SFTPC p.Lys63Glu and p.Ser72Asn/p.Gly100Ala), and one with ACD/MPV (FOXF1 p.Leu300ArgfsX79). None showed SFTPB mutations or defects in pSTAT-5. The 17 bronchoalveolar lavage or tracheal aspirates contained enough SP-B protein. CONCLUSION: The SP-C abnormality was most prevalent, and SP-B deficiency was rare in Japanese infants with hereditary ILD.

Pulmonary haptoglobin (pHp) is part of the surfactant system in the human lung.

Since the existence of pHp was demonstrated, it has been shown that this molecule and its receptor CD163 are regulated by different stimuli. Furthermore, a comparably fast secretion of pHp was described as well as the immuno-stimulatory effects. The intention of this study was to elucidate the role of pHp in the human lungs further. Here we show, by means of confocal microscopy and immune-electron-microscopy, a clear co-localization of pHp with surfactant protein-B in lamellar bodies of alveolar epithelial cells type II. These results are underlined by immunohistochemical stainings in differently fixed human lung tissues, which show pHp in vesicular and released form. The images of the released form resemble the intended position of surfactant in the human alveolus. pHp is secreted by Alveolar epithelial cells type II as previously shown. Moreover, pHp is co-localized with Surfactant protein-B. We conclude that the presented data shows that pHp is a native part of the surfactant system in the human lung. VIRTUAL SLIDES: http://www.diagnosticpathology.diagnomx.eu/vs/2563584738239912.

The alveolar epithelial differentiation of glandular inner lining cells in a mucoepidermoid carcinoma of the lung: a case report.

Mucoepidermoid carcinoma is a common malignant epithelial tumor of salivary glands, but relatively rare in lung. The histological features of mucoepidermoid carcinoma of the lung are similar to its counterpart arising from the salivary glands. Here, we reported a special tumor that occurred in the medial segment of the right lower lobe in a 22-year-old man. This tumor exhibited typical features of mucoepidermoid carcinoma with 3 cell types: squamoid cells, mucin-secreting cells and cells of intermediate type. These 3 types of cells organized into cysts, nests, glands and solid patterns. Specially, the inner lining cells of some glandular structures were uniform cuboidal and hobnail-like, similar to the alveolar epithelial cells. Immunohistochemistry staining revealed that the inner lining cells of glandular structures were positive for thyroid transcription factor-1 and surfactant protein-B, used as markers of alveolar epithelial cells, and were negative for p63. These findings for the first time demonstrated a rare alveolar epithelial differentiation of glandular inner lining cells in a mucoepidermoid carcinoma of the lung. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/7095988968057804.

[Effect of hyperoxia and TGF-ß1 on epithelial-mesenchymal transition of type II alveolar epithelial cells].

AIM: To investigate the effect of hyperoxia and TGF-ß1 on epithelial-mesenchymal transition (EMT)of type II alveolar epithelial cells (AEC-II) of mice. METHODS: AEC-II cells (MLE-12 lines) were randomly divided into following groups: air exposure group, hyperoxia exposure group, air exposure combined with TGF-ß1 treatment group, hyperoxia exposure combined with TGF-ß1 treatment group. The morphological changes of cells in each group were observed at 6, 12, 24, 48 hours. The protein and mRNA expressions of AECII specific marker lung surfactant protein B(SP-B)and fibroblast specific marker fibroblast specific protein (FSP1)were detected by double-labeled immunofluorescence and real time-PCR at the same time point, respectively. RESULTS: Along with the time of exposure to hyperoxia and TGF-ß1, AECIIcells gradually changed from pebble-like shape to spindle shape, and showed some fibroblast appearances. Synchronously, the protein expression of SP-B in AECII cells decreased, whereas the expression of FSP1 increased. The co-expressed were observed at 24 hours. Comparing with that of the air exposure group, the mRNA expression of SP-B in the hyperoxia exposure group, air exposure combined with TGF-ß1 treatment group, hyperoxia exposure combined with TGF-ß1 treatment group decreased significantly, whereas the mRNA expression of FSP1 increased significantly at 24 hours and 48 hours (P<0.01). CONCLUSION: Hyperoxia and TGF-ß1 can induce EMT of type II alveolar epithelial cells in a time-dependent manner.

The development of selected reaction monitoring methods for targeted proteomics via empirical refinement.

Software advancements in the last several years have had a significant impact on proteomics from method development to data analysis. Herein, we detail a method, which uses our in-house developed software tool termed Skyline, for empirical refinement of candidate peptides from targeted proteins. The method consists of four main steps from generation of a testable hypothesis, method development, peptide refinement, to peptide validation. The ultimate goal is to identify the best performing peptide in terms of ionization efficiency, reproducibility, specificity, and chromatographic characteristics to monitor as a proxy for protein abundance. It is important to emphasize that this method allows the user to perform this refinement procedure in the sample matrix and organism of interest with the instrumentation available. Finally, the method is demonstrated in a case study to determine the best peptide to monitor the abundance of surfactant protein B in lung aspirates.

A method to determine the kinetics of multiple proteins in human infants with respiratory distress syndrome.

We report a method to measure in vivo turnover of four proteins from sequential tracheal aspirates obtained from human newborn infants with respiratory distress syndrome using targeted proteomics. We detected enrichment for all targeted proteins approximately 3 h from the start of infusion of [5,5,5-(2)H(3)] leucine, secretion times that varied from 1.2 to 2.5 h, and half lives that ranged between 10 and 21 h. Complement factor B, a component of the alternative pathway of complement activation, had an approximately twofold-longer half-life than the other three proteins. In addition, the kinetics of mature and carboxy-terminal tryptic peptides from the same protein (surfactant protein B) were not statistically different (p = 0.49).

Runx3 is required for the differentiation of lung epithelial cells and suppression of lung cancer.

Human lung adenocarcinoma, the most prevalent form of lung cancer, is characterized by many molecular abnormalities. K-ras mutations are associated with the initiation of lung adenocarcinomas, but K-ras-independent mechanisms may also initiate lung tumors. Here, we find that the runt-related transcription factor Runx3 is essential for normal murine lung development and is a tumor suppressor that prevents lung adenocarcinoma. Runx3-/- mice, which die soon after birth, exhibit alveolar hyperplasia. Importantly, Runx3-/- bronchioli exhibit impaired differentiation, as evidenced by the accumulation of epithelial cells containing specific markers for both alveolar (that is SP-B) and bronchiolar (that is CC10) lineages. Runx3-/- epithelial cells also express Bmi1, which supports self-renewal of stem cells. Lung adenomas spontaneously develop in aging Runx3+/- mice ( approximately 18 months after birth) and invariably exhibit reduced levels of Runx3. As K-ras mutations are very rare in these adenomas, Runx3+/- mice provide an animal model for lung tumorigenesis that recapitulates the preneoplastic stage of human lung adenocarcinoma development, which is independent of K-Ras mutation. We conclude that Runx3 is essential for lung epithelial cell differentiation, and that downregulation of Runx3 is causally linked to the preneoplastic stage of lung adenocarcinoma.

Measurement of human surfactant protein-B turnover in vivo from tracheal aspirates using targeted proteomics.

We describe a method to measure protein synthesis and catabolism in humans without prior purification and use the method to measure the turnover of surfactant protein-B (SP-B). SP-B, a lung-specific, hydrophobic protein essential for fetal-neonatal respiratory transition, is present in only picomolar quantities in tracheal aspirate samples and difficult to isolate for dynamic turnover studies using traditional in vivo tracer techniques. Using infusion of [5,5,5-(2)H(3)] leucine and a targeted proteomics method, we measured both the quantity and kinetics of SP-B tryptic peptides in tracheal aspirate samples of symptomatic newborn infants. The fractional synthetic rate (FSR) of SP-B measured using the most abundant proteolytic fragment, a 10 amino acid peptide from the carboxy-terminus of proSP-B (SPTGEWLPR), from the circulating leucine pool was 0.035 +/- 0.005 h(-1), and the fractional catabolic rate was 0.044 +/- 0.003 h(-1). This technique permits high-throughput and sensitive measurement of turnover of low abundance proteins with minimal sample preparation.

Role of vagal innervation on pulmonary surfactant system during fetal development.

Vagally mediated afferent feedback and compliant lungs (surfactant system) play vital roles in the establishment of adequate alveolar ventilation and pulmonary gas exchange at birth. Although the significance of vagal innervation in the establishment of normal breathing patterns is well recognized, the precise role of lung innervation in the maturation of the surfactant system remains unclear. The specific aim of the present study was to investigate whether vagal denervation compromises the surfactant system during fetal development. Experiments were performed on 12 time-dated fetal sheep: 8 underwent cervical vagal denervation, and 4 were sham operated. Vagal denervation was performed at 110-113 days gestation. Fetal lambs were instrumented in utero to record arterial pH and blood-gas tensions. The animals were delivered by cesarean section under general anesthesia between 130 and 133 days gestation (term approximately 147 days). Lung samples were collected for wet-to-dry ratios, light and electron microscopy, and overall lung morphology. In addition, total proteins, total phospholipids, and surfactant proteins A and B were analyzed in both lung tissue and bronchoalveolar lavage fluid. Vagal denervation had no effect on alveolar architecture, including type II cells or the morphology of lamellar bodies within them. Furthermore, surfactant proteins A and B and total phospholipids were similar in lung tissue and bronchoalveolar lavage fluid between the two groups. A significant correlation was observed between circulating cortisol concentrations and surfactant proteins in the bronchoalveolar lavage fluid and lung tissue. We provide definitive evidence that vagal innervation at midgestation is not required for maturation of the pulmonary surfactant system during fetal development.

Pulmonary surfactant protein B in the peripheral circulation and functional impairment in patients with chronic heart failure.

INTRODUCTION AND OBJECTIVES: Surfactant protein B (SP-B) is a marker of damage to the alveolar-capillary barrier that could be useful for monitoring functional impairment in patients with chronic heart failure (HF). METHODS: Dyspnea-limited cardiopulmonary exercise testing was carried out in 43 outpatients with chronic HF (age 51+/-10 years, 77% male, left ventricular ejection fraction [LVEF] 33+/-11%). Peripheral blood serum samples were obtained at rest and during the first minute of peak exercise. The presence and concentration of SP-B in the serum samples were determined by Western blot analysis. RESULTS: At rest, SP-B was detected in 35 (82%) patients compared with only six (23%) healthy volunteers in a control group (n=26, age 51+/-10 years, 77% male). The median circulating SP-B level was higher in HF patients, at 174 [interquartile range, 70-283] vs. 77 [41-152] (P< .001) in the control group. In HF patients, the presence of circulating SP-B was associated with a lower LVEF (31.4+/-9.6% vs. 41.8+/-15%; P=.01). Multivariate analysis showed that the resting SP-B level correlated with a greater VE/VCO2 slope (beta=1.45; P=.02). The peak-exercise SP-B level correlated almost perfectly with the resting level (r=0.980; P< .001), but there was no significant increase with exercise (P=.164). Nor was there a correlation with any other exercise parameter. CONCLUSIONS: In patients with chronic HF, the level of pulmonary surfactant protein B in the peripheral circulation is increased and is correlated with ventilatory inefficiency during exercise, as indicated by the VE/VCO2 slope.

Choosing a right surfactant for respiratory distress syndrome treatment.

Respiratory distress syndrome (RDS) is the most common cause of respiratory insufficiency in preterm infants, especially those born at <30 weeks of gestation. Continuous positive airway pressure has been used since the 1970s as a primary mode of treatment for RDS. Surfactant therapy became available in the 1980s and has become the standard care for infants with or at risk for RDS. Surfactant therapy has been shown to decrease air leaks, neonatal and infant mortality as well as cost among survivors. Natural surfactants derived from animal sources containing surfactant proteins B (SP-B) and C (SP-C) as well as synthetic surfactants with functional SP-B- or SP-C-like protein mimics have been extensively evaluated in preterm neonates with or at risk for RDS. Evidence from randomized controlled trials indicates that treatment with natural surfactants results in faster weaning of supplemental oxygen and mean airway pressure, decreased duration of mechanical ventilation, and decreased mortality when compared to synthetic surfactants. Furthermore, at the present time, there are no approved synthetic surfactants available for use in preterm infants. Beractant, calfactant and poractant alpha are the three commonly used natural surfactants worldwide. Comparative studies including prospective randomized trials as well as large retrospective studies have shown significant differences in outcome and cost among these three natural surfactants. Of the eight prospective, randomized controlled trials and two retrospective studies involving the natural surfactant preparations, treatment with poractant alpha resulted in a significantly decreased mortality, decreased need for additional doses, faster weaning of oxygen and reduced hospital costs when compared to treatment with beractant or calfactant. These differences in outcome may be due to differences in phospholipid and SP-B content, amount of antioxidant phospholipids, plasmalogens, anti-inflammatory properties and viscosity among these three surfactants. Additional studies of administering surfactant non-invasively via laryngeal mask airway in preterm infants weighing >1,200 g and as an aerosol preparation are currently in progress.

Effect of inhaled budesonide on surfactant protein expression in asthmatic mice.

Pulmonary surfactant dysfunction may significantly contribute to small airway obstruction during the asthmatic response. Inhaled corticosteroids have been routinely used in the treatment of asthma, but neither its exact role nor its regulation on pulmonary surfactant is completely understood. The aim of this study was to determine the effect of budesonide on surfactant protein (SP) expression in asthmatic mice. Moreover, we investigated the function of transforming growth factor (TGF) beta signaling in the pulmonary surfactant system to identify a novel target for the treatment of asthma. Mice were sensitized and challenged by ovalbumin (OVA) to establish a murine model of asthma. To assess the effect of budesonide on asthmatic mice, animals were treated with aerosolized budesonide before OVA challenge. The levels of TGF-beta(1) in bronchoalveolar lavage fluid were analyzed by ELISA. The expressions of TGF-beta type I receptor, TGF-beta type II receptor, Smad2/3, Smad4, Smad7, and SPs were measured by immunochemistry technique and computerized image analysis system. SP-A and SP-B were significantly decreased after allergic sensitization and challenge, accompanied by active TGF-beta/Smad signal transduction. A dramatic increase in the expressions of SP-A and SP-B was observed after budesonide treatment. This was also accompanied by up-regulation of Smad7 expression and down-regulation of TGF-beta type I receptor expression. A possible explanation for the result is that an early budesonide inhaled treatment inhibits TGF-beta-induced reduction of SP-A and SP-B expression through inhibition of active TGF-beta/Smad signal transduction pathway in asthmatic mice. TGF-beta signaling may be a potentially important therapeutic target for antiasthma drugs.

[Novel procedure for preoperative diagnosis of papillary thyroid carcinoma].

Our study has shown that evaluation of marker genes SFTPB and TFF3 expression using fine needle aspiration biopsy of thyroid nodal alterations is an effective means of differential diagnosis of papillary thyroid carcinoma. When used on molecular level, it may detect the disease before clinical signs develop or ultrasound examination is carried out.

KL4-surfactant prevents hyperoxic and LPS-induced lung injury in mice.

KL(4)-surfactant contains the novel KL(4) peptide, sinapultide, which mimics properties of the hydrophobic pulmonary surfactant protein SP-B, in a phospholipid formulation and may be lung protective in experimental acute respiratory distress syndrome/acute lung injury. Our objective was to determine the protective role of airway delivery of KL(4)-surfactant in murine models of hyperoxic and lipopolysaccharide (LPS)-induced lung injury and further explore the mechanisms of protection. For the hyperoxic injury model, mice exposed to 80% O(2) for 6 days received an intranasal bolus of vehicle, beractant, or KL(4)-surfactant on days 3, 4, 5, and 6 of the exposure, and lungs were evaluated on day 7. Mice in the LPS-induced lung injury model received an intratracheal bolus of LPS followed by an intranasal bolus of KL(4)-surfactant or control at 1, 3, and 19 hr post-LPS challenge, and lungs were evaluated after 24 hr. To explore the mechanisms of protection, in vitro assays were performed with human and murine endothelial cell monolayers, and polymorphonuclear leukocyte (PMN) transmigration in the presence or absence of KL(4)-surfactant or lipid controls was evaluated. Based on morphology, histopathology, white blood cell count, percentage of PMNs, and protein concentration in bronchoalveolar lavage fluid, our data showed KL(4)-surfactant, unlike vehicle or beractant, blocked neutrophil influx into alveoli and suppressed lung injury. Furthermore, in vitro assays showed KL(4)-surfactant decreased neutrophil transmigration at the endothelial cell level. KL(4)-surfactant decreased inflammation and lung permeability compared with controls in both mouse models of lung injury. Evidence suggests the anti-inflammatory mechanism of the KL(4)-peptide is through inhibition of PMN transmigration through the endothelium.

Micelles of pulmonary surfactant in human amniotic fluid at term.

Studies using in vitro analysis have shown that the interaction between pulmonary surfactant and vernix caseosa could explain the appearance of amniotic fluid turbidity. That phenomenon is interpreted based on the "roll-up" hypothesis. We tested the roll-up hypothesis by examining the presence of micelles of pulmonary surfactant in human amniotic fluid at term. Amniotic fluid samples were collected from each of six healthy pregnant women at term and at 16 wk of gestation. These samples were stained negatively and analyzed using an electron microscope. Ultrastructures present in amniotic fluid were compared with the structure of micelles derived from suspended surfactant TA isolated from bovine lung. Surfactant TA formed spheroidal and rod-shaped micelles 10-70 nm in diameter above the critical micelle concentration. Identical micelle particles were described in human amniotic fluid at term. In addition, surfactant protein B was identified in the micelle fraction of amniotic fluid. However, no micelles were found in human amniotic fluid taken at 16 wk of gestation. Our results support the view that pulmonary surfactant could induce the detachment of vernix caseosa and increase the turbidity of the amniotic fluid.

Lipid specificity of surfactant protein B studied by time-of-flight secondary ion mass spectrometry.

One of the key functions of mammalian pulmonary surfactant is the reduction of surface tension to minimal values. To fulfill this function it is expected to become enriched in dipalmitoylphosphatidylcholine either on its way from the alveolar type II pneumocytes to the air/water interface of the lung or within the surface film during compression and expansion of the alveoli during the breathing cycle. One protein that may play a major role in this enrichment process is the surfactant protein B. The aim of this study was to identify the lipidic interaction partner of this protein. Time-of-flight secondary ion mass spectrometry was used to analyze the lateral distribution of the components in two SP-B-containing model systems. Either native or partly isotopically labeled lipids were analyzed. The results of both setups give strong indications that, at least under the specific conditions of the chosen model systems (e.g., concerning pH and lipid composition), the lipid interacting with surfactant protein B is not phosphatidylglycerol as generally accepted, but dipalmitoylphosphatidylcholine instead.