Disaturated Phosphatidylcholine Research Articles

A novel deuterium-based model for measurement of exogenous surfactant using deuterium-depleted water.

Stable isotope tracers, like 13 C, can be used for the measurement of the partition between the endogenous and exogenous pulmonary disaturated-phosphatidylcholine (DSPC). Deuterium labeling methods are still not fully explored. Our aim was to investigate the feasibility of using deuterium-depletedwater (DDW) and deuterium-enriched water (DEW) to measure endogenous and exogenous pulmonary DSPC in a rabbit model of surfactant depletion. Data obtained from the 13 C dilution method were used as a reference. We studied 9 adult rabbits: 4 drank DDW and 5 DEW for 5 days. Lung surfactant depletion was induced at Day 5 by repeated saline bronchoalveolar lavages (BAL), which were stored as a pool (BAL pool). After endogenous surfactant depletion, rabbits received exogenous surfactant followed by a second BAL depletion procedure (End-Experiment Pool). DSPC quantity, and palmitic acid (PA)-DSPC 2 H/1 H (δ2 H) and 13 C/12 C ratios (δ13 C) of exogenous surfactant batches and of BAL pools were measured by High-Resolution Mass Spectrometry. The amount of exogenous surfactant recovered from the lungs ranged from 45% to 81% and, it was highly correlated with those obtained with the use of the 13 C (r = 0.9844, p < 0.0001). We demonstrated that commercially available purified DDW and even low doses of DEW can be used to modify the deuterium background of endogenous surfactants with the purpose of measuring the contribution of exogenous surfactants to the endogenous alveolar surfactant pool.

Surfactant lung delivery with LISA and InSurE in adult rabbits with respiratory distress.

BackgroundIn preterm infants, InSurE (Intubation–Surfactant–Extubation) and LISA (less invasive surfactant administration) techniques allow for exogenous surfactant administration while reducing lung injury associated with mechanical ventilation. We compared the acute pulmonary response and lung deposition of surfactant by LISA and InSurE in surfactant-depleted adult rabbits.MethodsTwenty-six spontaneously breathing surfactant-depleted adult rabbits (6–7 weeks old) with moderate RDS and managed with nasal continuous positive airway pressure were randomized to 3 groups: (1) 200 mg/kg of surfactant by InSurE; (2) 200 mg/kg of surfactant by LISA; (3) no surfactant treatment (Control). Gas exchange and lung mechanics were monitored for 180 min. After that, surfactant lung deposition and distribution were evaluated monitoring disaturated-phosphatidylcholine (DSPC) and surfactant protein C (SP-C), respectively.ResultsNo signs of recovery were found in the untreated animals. After InSurE, oxygenation improved more rapidly compared to LISA. However, at 180’ LISA and InSurE showed comparable outcomes in terms of gas exchange, ventilation parameters, and lung mechanics. Neither DSPC in the alveolar pool nor SP-C signal distributions in a frontal lung section were significantly different between InSurE and LISA groups.ConclusionsIn an acute setting, LISA demonstrated efficacy and surfactant lung delivery similar to that of InSurE in surfactant-depleted adult rabbits.Impact Although LISA technique is gaining popularity, there are still several questions to address. This is the first study comparing LISA and InSurE in terms of gas exchange, ventilation parameters, and lung mechanics as well as surfactant deposition and distribution.In our animal study, three hours post-treatment, LISA method seems to be as effective as InSurE and showed similar surfactant lung delivery.Our findings provide some clarifications on a fair comparison between LISA and InSurE techniques, particularly in terms of surfactant delivery. They should reassure some of the concerns raised by the clinical community on LISA adoption in neonatal units.

Tracing exogenous surfactant in vivo in rabbits by the natural variation of 13C

BackgroundRespiratory Distress Syndrome (RDS) is a prematurity-related breathing disorder caused by a quantitative deficiency of pulmonary surfactant. Surfactant replacement therapy is effective for RDS newborns, although treatment failure has been reported. The aim of this study is to trace exogenous surfactant by 13C variation and estimate the amount reaching the lungs at different doses of the drug.MethodsForty-four surfactant-depleted rabbits were obtained by serial bronchoalveolar lavages (BALs), that were merged into a pool (BAL pool) for each animal. Rabbits were in nasal continuous positive airway pressure and treated with 0, 25, 50, 100 or 200 mg/kg of poractant alfa by InSurE. After 90 min, rabbits were depleted again and a new pool (BAL end experiment) was collected. Disaturated-phosphatidylcholine (DSPC) was measured by gas chromatography. DSPC-Palmitic acid (PA) 13C/12C was analyzed by isotope ratio mass spectrometry. One-way non-parametric ANOVA and post-hoc Dunn’s multiple comparison were used to assess differences among experimental groups.ResultsBased on DSPC-PA 13C/12C in BAL pool and BAL end experiment, the estimated amount of exogenous surfactant ranged from 61 to 87% in dose-dependent way (p < 0.0001) in animals treated with 25 up to 200 mg/kg. Surfactant administration stimulated endogenous surfactant secretion. The percentage of drug recovered from lungs did not depend on the administered dose and accounted for 31% [24–40] of dose.ConclusionsWe reported a risk-free method to trace exogenous surfactant in vivo. It could be a valuable tool for assessing, alongside the physiological response, the delivery efficiency of surfactant administration techniques.

From bench to bedside: in vitro and in vivo evaluation of a neonate-focused nebulized surfactant delivery strategy

BackgroundNon-invasive delivery of nebulized surfactant has been a neonatology long-pursued goal. Nevertheless, the clinical efficacy of nebulized surfactant remains inconclusive, in part, due to the great technical challenges of depositing nebulized drugs in the lungs of preterm infants. The aim of this study was to investigate the feasibility of delivering nebulized surfactant (poractant alfa) in vitro and in vivo with an adapted, neonate-tailored aerosol delivery strategy.MethodsParticle size distribution of undiluted poractant alfa aerosols generated by a customized eFlow-Neos nebulizer system was determined by laser diffraction. The theoretical nebulized surfactant lung dose was estimated in vitro in a clinical setting replica including a neonatal continuous positive airway pressure (CPAP) circuit, a cast of the upper airways of a preterm neonate, and a breath simulator programmed with the tidal breathing pattern of an infant with mild respiratory distress syndrome (RDS). A dose-response study with nebulized surfactant covering the 100–600 mg/kg nominal dose-range was conducted in RDS-modelling, lung-lavaged spontaneously-breathing rabbits managed with nasal CPAP. The effects of nebulized poractant alfa on arterial gas exchange and lung mechanics were assessed. Exogenous alveolar disaturated-phosphatidylcholine (DSPC) in the lungs was measured as a proxy of surfactant deposition efficacy.ResultsLaser diffraction studies demonstrated suitable aerosol characteristics for inhalation (mass median diameter, MMD = 3 μm). The mean surfactant lung dose determined in vitro was 13.7% ± 4.0 of the 200 mg/kg nominal dose. Nebulized surfactant delivered to spontaneously-breathing rabbits during nasal CPAP significantly improved arterial oxygenation compared to animals receiving CPAP only. Particularly, the groups of animals treated with 200 mg/kg and 400 mg/kg of nebulized poractant alfa achieved an equivalent pulmonary response in terms of oxygenation and lung mechanics as the group of animals treated with instilled surfactant (200 mg/kg).ConclusionsThe customized eFlow-Neos vibrating-membrane nebulizer system efficiently generated respirable aerosols of undiluted poractant alfa. Nebulized surfactant delivered at doses of 200 mg/kg and 400 mg/kg elicited a pulmonary response equivalent to that observed after treatment with an intratracheal surfactant bolus of 200 mg/kg. This bench-characterized nebulized surfactant delivery strategy is now under evaluation in Phase II clinical trial (EUDRACT No.:2016–004547-36).

Hepatic Steatosis Accompanies Pulmonary Alveolar Proteinosis.

Maintenance of tissue-specific organ lipid compositions characterizes mammalian lipid homeostasis. The lungs and liver synthesize mixed phosphatidylcholine (PC) molecular species that are subsequently tailored for function. The lungs progressively enrich disaturated PC directed to lamellar body surfactant stores before secretion. The liver accumulates polyunsaturated PC directed to very-low-density lipoprotein assembly and secretion, or to triglyceride stores. In each tissue, selective PC species enrichment mechanisms lie at the heart of effective homeostasis. We tested for potential coordination between these spatially separated but possibly complementary phenomena under a major derangement of lung PC metabolism, pulmonary alveolar proteinosis (PAP), which overwhelms homeostasis and leads to excessive surfactant accumulation. Using static and dynamic lipidomics techniques, we compared (1) tissue PC compositions and contents, and (2) in lungs, the absolute rates of synthesis in both control mice and the granulocyte-macrophage colony-stimulating factor knockout model of PAP. Significant disaturated PC accumulation in bronchoalveolar lavage fluid, alveolar macrophage, and lavaged lung tissue occurred alongside increased PC synthesis, consistent with reported defects in alveolar macrophage surfactant turnover. However, microscopy using oil red O staining, coherent anti-Stokes Raman scattering, second harmonic generation, and transmission electron microscopy also revealed neutral-lipid droplet accumulations in alveolar lipofibroblasts of granular macrophage colony-stimulating factor knockout animals, suggesting that lipid homeostasis deficits extend beyond alveolar macrophages. PAP plasma PC composition was significantly polyunsaturated fatty acid enriched, but the content was unchanged and hepatic polyunsaturated fatty acid-enriched PC content increased by 50% with an accompanying micro/macrovesicular steatosis and a fibrotic damage pattern consistent with nonalcoholic fatty liver disease. These data suggest a hepatopulmonary axis of PC metabolism coordination, with wider implications for understanding and managing lipid pathologies in which compromise of one organ has unexpected consequences for another.

Neonatal Respiratory Diseases in the Newborn Infant: Novel Insights from Stable Isotope Tracer Studies

Respiratory distress syndrome is a common problem in preterm infants and the etiology is multifactorial. Lung underdevelopment, lung hypoplasia, abnormal lung water metabolism, inflammation, and pulmonary surfactant deficiency or disfunction play a variable role in the pathogenesis of respiratory distress syndrome. High-quality exogenous surfactant replacement studies and studies on surfactant metabolism are available; however, the contribution of surfactant deficiency, alteration or dysfunction in selected neonatal lung conditions is not fully understood. In this article, we describe a series of studies made by applying stable isotope tracers to the study of surfactant metabolism and lung water. In a first set of studies, which we call ‘endogenous studies', using stable isotope-labelled intravenous surfactant precursors, we showed the feasibility of measuring surfactant synthesis and kinetics in infants using several metabolic precursors including plasma glucose, plasma fatty acids and body water. In a second set of studies, named ‘exogenous studies', using stable isotope-labelled phosphatidylcholine tracer given endotracheally, we could estimate surfactant disaturated phosphatidylcholine pool size and half-life. Very recent studies are focusing on lung water and on the endogenous biosynthesis of the surfactant-specific proteins. Information obtained from these studies in infants will help to better tailor exogenous surfactant treatment in neonatal lung diseases.

Does Whole-Body Hypothermia in Neonates with Hypoxic-Ischemic Encephalopathy Affect Surfactant Disaturated-Phosphatidylcholine Kinetics?

BackgroundIt is unknown whether Whole-Body Hypothermia (WBH) affects pulmonary function. In vitro studies, at relatively low temperatures, suggest that hypothermia may induce significant changes to the surfactant composition. The effect of WBH on surfactant kinetics in newborn infants is unknown. We studied in vivo kinetics of disaturated-phosphatidylcholine (DSPC) in asphyxiated newborns during WBH and in normothermic controls (NTC) with no or mild asphyxia. Both groups presented no clinically apparent lung disease.MethodsTwenty-seven term or near term newborns requiring mechanical ventilation were studied (GA 38.6±2.2 wks). Fifteen during WBH and twelve NTC. All infants received an intra-tracheal dose of 13C labelled DSPC and tracheal aspirate were performed. DSPC amount, DSPC half-life (HL) and pool size (PS) were calculated.ResultsDSPC amount in tracheal aspirates was 0.42 [0.22–0.54] and 0.36 [0.10–0.58] mg/ml in WBH and NTC respectively (p = 0.578). DSPC HL was 24.9 [15.7–52.5] and 25.3 [15.8–59.3] h (p = 0.733) and DSPC PS was 53.2 [29.4–91.6] and 40.2 [29.8–64.6] mg/kg (p = 0.598) in WBH and NTC respectively.ConclusionsWBH does not alter DSPC HL and PS in newborn infants with no clinical apparent lung disease.

Surfactant protein B and A concentrations are increased in neonatal pneumonia.

Background:Term newborns with pneumonia show a reduced pulmonary compliance due to multiple and ill-defined factors. Surfactant proteins’ (SPs) changes could have a role in the reduced compliance but the matter is still unsettled. The aim of this study was to clarify the meaning of SPs changes during pneumonia in term newborns.Methods:In 28 term ventilated newborns, 13 with pneumonia and 15 with no lung disease, we measured SP-B, SP-A, disaturated-phosphatidylcholine (DSPC), and total phospholipids (PL) concentrations in tracheal aspirates at intubation and close to extubation. We also measured DSPC kinetics using (U-13C-PA)dipalmitoyl-phosphatidylcholine.Results:At baseline, SP-B, expressed as % of PL, was significantly different between the groups, being 3.5-fold higher in pneumonia than controls. Conversely, SP-A did not vary between the groups. At extubation, SP-B and SP-A concentrations had decreased significantly in newborns with pneumonia, while there was no significant change in controls. DSPC t1/2 was significantly shorter in the pneumonia group (11.8 (5.5–19.8) h vs. 26.6 (19.3–63.6) h, P = 0.011).Conclusion:In term newborns with pneumonia, SP-B increases with respect to PL, and DSPC is turned over at a faster rate. Disease’s resolution is associated with the restoration of the normal ratio between SP-B and PL.Supplementary informationThe online version of this article (doi:10.1038/pr.2015.123) contains supplementary material, which is available to authorized users.

Serine 32 to threonine mutation preserves peroxiredoxin 6 structure and enzymatic function but abolishes its trafficking to lamellar bodies in vivo

Peroxiredoxin 6 (Prdx6), a bifunctional protein with lysosomal‐type phospholipase A2 (aiPLA2) and GSH peroxidase activities, protects lungs from oxidative stress and participates in lung surfactant phospholipid (PL) turnover. Prdx6 has been localized to both cytosol and acidic organelles (lamellar bodies [LB] and lysosomes) in lung epithelium and its organellar targeting sequence has been identified (31DSWGILFSHP40, Am.J.Physiol.297 : L871, 2009). Interestingly, an S32T mutation fully preserved secondary structure of recombinant protein and had no effect on either its peroxidase or aiPLA2 activity. In a “knock‐in” mouse model carrying the S32T mutation in the Prdx6 gene, Prdx6 protein and aiPLA2 activity were present in lung epithelial cells. However,the protein was absent from epithelial cell LB. Lung surfactant dipalmitoylphosphatidylcholine (DPPC) is endocytosed by alveolar epithelial cells and degraded by aiPLA2. The content of total phospholipid, PC, and disaturated phosphatidylcholine (DSPC) in bronchoalveolar lavage fluid, lung lamellar bodies, and lung homogenate was increased in S32T mutant animals and degradation of internalized [3H]DPPC in isolated mouse lungs was significantly decreased. Thus, Thr can substitute for Ser for the enzymatic activities of Prdx6 but not for its targeting to LB. These results confirm an important role for lamellar body Prdx6 in the degradation and remodeling of lung surfactant phosphatidylcholine. [HL‐019737, HL102016 and HL105509]

In vivo effect of pneumonia on surfactant disaturated-phosphatidylcholine kinetics in newborn infants.

BackgroundBacterial pneumonia in newborns often leads to surfactant deficiency or dysfunction, as surfactant is inactivated or its production/turnover impaired. No data are available in vivo in humans on the mechanism of surfactant depletion in neonatal pneumonia. We studied the kinetics of surfactant's major component, disaturated-phosphatidylcholine (DSPC), in neonatal pneumonia, and we compared our findings with those obtained from control newborn lungs.MethodsWe studied thirty-one term or near-term newborns (gestational age 39.7±1.7 weeks, birth weight 3185±529 g) requiring mechanical ventilation. Fifteen newborns had pneumonia, while 16 newborns were on mechanical ventilation but had no lung disease. Infants received an intratracheal dose of 13C labeled dipalmitoyl-phosphatidylcholine at the study start. We measured the amount and the isotopic enrichment of DSPC-palmitate from serial tracheal aspirates by gas chromatography and gas chromatography-mass spectrometry, respectively, and we calculated the DSPC half-life (HL) and pool size (PS) from the isotopic enrichment curves of surfactant DSPC-palmitate.ResultsThe mean DSPC amount obtained from all tracheal aspirates did not differ between the two groups. DSPC HL was 12.7 (6.5–20.2) h and 25.6 (17.9–60.6) h in infants with pneumonia compared with control infants (p = 0.003). DSPC PS was 14.1 (6.6–30.9) mg/kg in infants with pneumonia and 34.1 (25.6–65.0) mg/kg in controls, p = 0.042. Myeloperoxidase (MPO) activity, as a marker of lung inflammation, was 1322 (531–2821) mU/ml of Epithelial Lining Fluid (ELF) and 371(174–1080) mU/ml ELF in infants with pneumonia and in controls, p = 0.047. In infants with pneumonia, DSPC PS and HL significantly and inversely correlated with mean Oxygenation Index (OI) during the study (DSPC PS vs. OI R = −0.710, p = 0.004 and HL vs. OI R = −0.525, p = 0.044, respectively).ConclusionsWe demonstrated for the first time in vivo in humans that DSPC HL and PS were markedly impaired in neonatal pneumonia and that they inversely correlated with the degree of respiratory failure.

Pulmonary surfactant synthesis after unilateral lung injury in mice

Aspiration pneumonitis can lead to alveolar surfactant dysfunction. We employed a murine model of unilateral aspiration to compare surfactant synthesis in the injured (I) and noninjured (NI) contralateral lung. Mice were instilled with hydrochloric acid in the right bronchus and, after 18 h, an intraperitoneal dose of deuterated water was administered as precursor of disaturated phosphatidylcholine (DSPC)-palmitate. Selected bronchoalveolar lavage fluid (BALF) was collected at scheduled time points and lungs were removed. We measured DSPC-palmitate synthesis in lung tissue and secretion in BALF by gas chromatography-isotope ratio mass spectrometry, together with total proteins and myeloperoxidase activity (MPO) by spectrophotometry. BALF total proteins and MPO were significantly increased in the I lungs compared with NI and naïve control lungs. The DSPC pool size was significantly lower in the BALF of the I lungs compared with naïve controls. DSPC synthesis was accelerated in the I and NI lungs. DSPC secretion of the I lungs was similar to their respective naïve controls, and it was markedly lower compared with their respective NI contralateral lungs. DSPC synthesis and secretion were faster, especially in the NI lungs, compared with naïve control lungs, as a possible compensatory mechanism due to a cross-talk between the lungs triggered by inflammation, hyperventilation, and/or undetermined type II cell reaction to the injury.

Impaired pulmonary defense against Pseudomonas aeruginosa in VEGF gene inactivated mouse lung

Repeated bacterial and viral infections are known to contribute to worsening lung function in several respiratory diseases, including asthma, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). Previous studies have reported alveolar wall cell apoptosis and parenchymal damage in adult pulmonary VEGF gene ablated mice. We hypothesized that VEGF expressed by type II cells is also necessary to provide an effective host defense against bacteria in part by maintaining surfactant homeostasis. Therefore, Pseudomonas aeruginosa (PAO1) levels were evaluated in mice following lung-targeted VEGF gene inactivation, and alterations in VEGF-dependent type II cell function were evaluated by measuring surfactant homeostasis in mouse lungs and isolated type II cells. In VEGF-deficient lungs increased PAO1 levels and pro-inflammatory cytokines, TNFα and IL-6, were detected 24 h after bacterial instillation compared to control lungs. In vivo lung-targeted VEGF gene deletion (57% decrease in total pulmonary VEGF) did not alter alveolar surfactant or tissue disaturated phosphatidylcholine (DSPC) levels. However, sphingomyelin content, choline phosphate cytidylyltransferase (CCT) mRNA, and SP-D expression were decreased. In isolated type II cells an 80% reduction of VEGF protein resulted in decreases in total phospholipids (PL), DSPC, DSPC synthesis, surfactant associated proteins (SP)-B and -D, and the lipid transporters, ABCA1 and Rab3D. TPA-induced DSPC secretion and apoptosis were elevated in VEGF-deficient type II cells. These results suggest a potential protective role for type II cell-expressed VEGF against bacterial initiated infection.

403 Surfactant Disaturated-Phosphatidylcholine Kinetics in Pneumonia by Stable Isotopes

Background and AimWhether pulmonary surfactant deficiency or dysfunction contributes to the pathogenesis of neonatal pneumonia is still debated. Aim of our study was to measure surfactant disaturated-phosphatidylcholine (DSPC) kinetics and...

Impaired surfactant protein B synthesis in infants with congenital diaphragmatic hernia

Pulmonary hypoplasia and hypertension account for significant morbidity and mortality in neonates with congenital diaphragmatic hernia (CDH). Whether CDH is associated with surfactant dysfunction remains controversial. Therefore, we measured disaturated phosphatidylcholine (DSPC) and surfactant protein (SP)-B concentration in tracheal aspirates and their synthesis rate in infants with CDH compared to infants without lung disease. (2)H2O as a precursor of DSPC and 1-(13)C-leucine as a precursor of SP-B were administered to 13 infants with CDH and eight controls matched for gestational age. DSPC and SP-B were isolated from tracheal aspirates, and their fractional synthesis rate was derived from (2)H and (13)C enrichment curves obtained by mass spectrometry. DSPC and SP-B amounts in tracheal aspirates were also measured. In infants with CDH, SP-B fractional synthesis rate and amount were 62±27% and 57±22% lower, respectively, than the value found in infants without lung disease (p<0.01 and p<0.05, respectively). There were no significant group differences in DSPC fractional synthesis rate and amount. Infants with CDH have a lower rate of synthesis of SP-B and less SP-B in tracheal aspirates. In these infants, partial SP-B deficiency could contribute to the severity of respiratory failure and its correction might represent a therapeutic goal.

Altered surfactant homeostasis and recurrent respiratory failure secondary to TTF-1 nuclear targeting defect

BackgroundMutations of genes affecting surfactant homeostasis, such as SFTPB, SFTPC and ABCA3, lead to diffuse lung disease in neonates and children. Haploinsufficiency of NKX2.1, the gene encoding the thyroid transcription factor-1 (TTF-1) - critical for lung, thyroid and central nervous system morphogenesis and function - causes a rare form of progressive respiratory failure designated brain-lung-thyroid syndrome. Molecular mechanisms involved in this syndrome are heterogeneous and poorly explored. We report a novel TTF-1 molecular defect causing recurrent respiratory failure episodes in an infant.MethodsThe subject was an infant with severe neonatal respiratory distress syndrome followed by recurrent respiratory failure episodes, hypopituitarism and neurological abnormalities. Lung histology and ultrastructure were assessed by surgical biopsy. Surfactant-related genes were studied by direct genomic DNA sequencing and array chromatine genomic hybridization (aCGH). Surfactant protein expression in lung tissue was analyzed by confocal immunofluorescence microscopy. For kinetics studies, surfactant protein B and disaturated phosphatidylcholine (DSPC) were isolated from serial tracheal aspirates after intravenous administration of stable isotope-labeled 2H2O and 13C-leucine; fractional synthetic rate was derived from gas chromatography/mass spectrometry 2H and 13C enrichment curves. Six intubated infants with no primary lung disease were used as controls.ResultsLung biopsy showed desquamative interstitial pneumonitis and lamellar body abnormalities suggestive of genetic surfactant deficiency. Genetic studies identified a heterozygous ABCA3 mutation, L941P, previously unreported. No SFTPB, SFTPC or NKX2.1 mutations or deletions were found. However, immunofluorescence studies showed TTF-1 prevalently expressed in type II cell cytoplasm instead of nucleus, indicating defective nuclear targeting. This pattern has not been reported in human and was not found in two healthy controls and in five ABCA3 mutation carriers. Kinetic studies demonstrated a marked reduction of SP-B synthesis (43.2 vs. 76.5 ± 24.8%/day); conversely, DSPC synthesis was higher (12.4 vs. 6.3 ± 0.5%/day) compared to controls, although there was a marked reduction of DSPC content in tracheal aspirates (29.8 vs. 56.1 ± 12.4% of total phospholipid content).ConclusionDefective TTF-1 signaling may result in profound surfactant homeostasis disruption and neonatal/pediatric diffuse lung disease. Heterozygous ABCA3 missense mutations may act as disease modifiers in other genetic surfactant defects.

Simultaneous measurement of phosphatidylglycerol and disaturated‐phosphatidylcholine palmitate kinetics from alveolar surfactant. Study in infants with stable isotope tracer, coupled with isotope ratio mass spectrometry

Disaturated-phosphatidylcholine (DSPC) and phosphatidylglycerol (PG) are respectively the first and the third most abundant phospholipid in human alveolar surfactant. Their concentration decreases in airway surfactant of adults and infants with respiratory distress syndrome and cystic fibrosis. In this study, we used mass spectrometry (IRMS) to investigate the turnover of DSPC and PG in tracheal aspirates (TA) obtained from infants with normal or diseased lungs. We studied eight infants requiring mechanical ventilation: two with no lung disease, four with diaphragmatic hernia, one with ATP-binding cassette sub-family A member 3 heterozygote mutation and one with sepsis. Patients received deuterated water for 48 h as metabolic precursors of palmitate-DSPC and palmitate-PG. Serial TAs were obtained every 6 h for five days or until extubation. DSPC and PG were isolated from TA by column and high-performance thin layer chromatography. Deuterium enrichments of palmitate-DSPC and PG residues were measured by IRMS coupled with a gas chromatographer. Median secretion time (ST), peak time (PT) and fractional synthesis rate (FSR) were 3.7 [0.9- 13.4] h, 71.0 [52.2 - 85.2] h and 6.6 [6.3 - 11.1] %/day for DSPC and 19.3 [6.4 - 22.8] h, 49.0 [33.0 - 52.5] h and 5.8 [4.8 - 10.9] %/day for PG. This study shows that it is feasible to use deuterium derived from body water to trace simultaneously airway surfactant DSPC and PG in humans. When compared within the same patient, DSPC and PG had similar fractional synthesis rates, but PG had a shorter PT, suggesting differences in the life cycle of these essential surfactant components.

Therapeutic role of terbutaline in a rat whole-lung lavage model

ABSTRACTWhole-lung lavage (WLL) is the preferred therapy for patients with pulmonary alveolar proteinosis (PAP). However, WLL can cause fluid retention, surfactant lost, and hypoxia. Terbutaline has been shown to accelerate the rate of alveolar fluid clearance and augment surfactant secretion. The present study aimed at investigating effects of terbutaline and oxygen ventilation in rats with WLL. Forty rats were randomly divided into control, ventilation, NS (normal saline) + ventilation, LT (terbutaline in low concentration, 10−4 M) + ventilation, and HT (terbutaline in high concentration, 10−3 M) + ventilation groups. The left thoracic cavity was opened and a cannula was inserted into the left bronchus. The left lung was ventilated, while the right lung was lavaged. Arterial blood gas, electrocardiogram, histological changes, and wet/dry ratio of lung tissues were examined. The concentrations of total protein (TP), total phospholipids (TPL), and disaturated phosphatidylcholine (DSPC) in recovery fluid were measured. For the in vitro study, alveolar type II (ATII) cells were isolated from healthy male rats, incubated for 24 hours, and divided into control, LT, and HT groups and exposed to different concentration of terbutaline (10−4 and 10−3 M) for 2 hours, followed by measuring sodium-potassium adenosine triphosphatase (Na+,K+-ATPase) activity. Oxygen ventilation significantly increased Pao2/Fio2 after lavage in the ventilation group, as compared to control group (249.4 ± 7.9 vs 210.6 ± 9.4; P = .001). Compared with NS + venlitation group, a higher concentration of terbutaline decreased the wet/dry ratio of lung tissues (5.0 ± 0.1 vs 5.6 ± 0.1; P = .007), increased the concentrations of TPL (175.9 ± 14.0 vs 162.0 ± 6.8 mg/L; P = .031) and DSPC (93.2 ± 6.9 vs 70.9 ± 6.2 mg/L, P = .0001) in the recovery fluid, and alleviated hypoxia significantly. Terbutaline in both low and high doses increased Na+,K+-ATPase activity in ATII cells (62.5 ± 2.4 and 62.6 ± 2.8 vs 32.2 ± 2.1 mmol/h/mg protein; P < .01). Theses results show that the administration of terbutaline facilitates alveolar fluid absorption and increases surfactant secretion during lung lavage, the former is partly driven by increasing Na+,K+-ATPase activity. The modified lavage method, with the use of terbutaline and oxygen ventilation, is one of potential therapies for patients with PAP.

The role of transforming growth factors beta1 and beta3 in pre- and post-natal pulmonary surfactant development

The aim was to explore the pulmonary surfactant regulatory effect of TGF-β1 and TGF-β3 during pre- and post-natal porcine development. Pigs on embryonic day 99 (E94) (term = 114 days) and 1-h (D0) and 15-day (D20) neonates were killed to obtain whole lungs. DSPC (disaturated phosphatidylcholine) was separated from other phospholipids, and chemical methods were used to determine the amounts of DSPC, TPL (total phospholipids) and TP (total protein) in BALF (bronchoalveolar lavage fluid). TPL was elevated at E94. DSPC in TPL was significantly higher in the D20 group than in the E94 group. Reductions in TP correlated with developmental age. The levels of TGF-β1 and TGF-β3 mRNA were determined by RT (reverse transcription)-PCR and Northern blot. The expression of TGF-β1 mRNA was low at E94, increased at D0 and then decreased at D20. The expression of TGF-β3 was high at E94, reduced at D0, and then elevated at D20. We further examined the effect of exogenously administered TGF-1 on the expression of SPs (surfactant proteins) and cytidine triphosphorylate: CCT (phosphocholine cytidylyltransferase) activity in porcine fetal lung cells cultured for 5 days. The results indicated that TGF-β1 inhibited the expression of all three SPs (SP-A, SP-B and SP-C) and CCT activity, but did not alter the expression level of SP-D transcripts. We conclude that TGF-1 inhibits the expression of surfactant components. The alterations of TGF-β3 seem to partly explain the pulmonary surfactant changes observed in development, but this result needs further investigation.

Pharmacokinetics and clinical predictors of surfactant redosing in respiratory distress syndrome

Limited data are available on predictors for surfactant retreatment in preterm infants with respiratory distress syndrome (RDS). To study the pharmacokinetics of exogenous surfactant and the clinical parameters associated with surfactant redosing. Exogenous surfactant pharmacokinetics was studied in 125 preterm infants (birth weight 997 ± 432 g; gestational age 28.0 ± 2.6 weeks) with moderate to severe RDS requiring mechanical ventilation. Clinical and respiratory parameters were recorded hourly, and surfactant disaturated-phosphatidylcholine (DSPC) half-life, pool size, and endogenous synthesis were calculated by stable isotope tracing of surfactant DSPC isolated from serial tracheal aspirates. Univariate and multiple logistic regression were used to study the effects of clinical and surfactant kinetic variables on the need for redosing. Fifty-three infants (42.4%) received one dose, 51 (40.8%) two doses, and 21 (16.8%) three doses. Median (interquartile range, IQR) DSPC half-life was 21 (13-39), 11 (7-17), and 10 (7-16) h after the first, second, and third dose, respectively (p = 0.07). Univariate analysis showed a significantly shorter DSPC half-life in infants requiring more surfactant doses. On logistic analysis, risk of redosing was higher with lower birth weight, worse radiological score, shorter DSPC half-life, and surfactant dose of 100 mg/kg, whilst it was lower with elective high-frequency ventilation at time of intubation, instead of conventional ventilation. When optimizing surfactant replacement therapy and its cost-benefit ratio, pharmacokinetics and clinical variables associated with need of redosing should be taken into account.

495 Impaired Surfactant Protein B Synthesis in Infants with Congenital Diaphragmatic Hernia

Background: Whether congenital diaphragmatic hernia (CDH) is associated with surfactant deficiency or dysfunction is controversial. Aims: To measure disaturated phosphatidylcholine (DSPC) and surfactant protein B (SP-B) synthesis and metabolism in infants with CDH. Methods: DSPC and SP-B amounts and kinetics were studied in tracheal aspirates (TA) of 12 infants with CDH (BW 2978±447g, GA 38±2 wk) and in 8 GA-matched control infants (BW 3160±350g, GA 38±2 wk). Seventeen infants received a 24h infusion of 13C-leucine, an i.v. bolus of 2H2O and 0.0625% of fluid intake as 2H2O every 12h over the next 48h. Three infants received only 13C-leucine infusion. DSPC and SP-B were isolated from TA. DSPC amounts, 2H-palmitate and 13C-leucine enrichments were measured by gas chromatography mass spectrometry. Results: DSPC amount and kinetics have been measured in 17 newborns, and SP-B kinetics in 18. DSPC median (interquartile range) fractional synthesis rate (FSR) was 5.0 %/day (3.5-8.5) in CDH patients vs 6.1 %/day (5.8-7.0) in controls (p=0.31). DSPC secretion time (ST) was 10.7h (6.9-11.5) vs 7.9h (5.2-14.0) (p=0.65). SP-B FSR was 33.4 %/day (28.4-42.9) vs 52.0 %/ day (40.0-83.4) (p=0.03); SP-B peak time was 26h (24-32) vs 18h (15-21) (p=0.02), both significantly lower in the CDH group. SP-B ST was 6.1h (4.0- 9.9) vs 2.7h (1.1-7.1) (p=0.24), and half life 7.5h (5.2-9.1) vs 11.1h (5.8-17.3) (p=0.33). Conclusions: Decreased and delayed SP-B synthesis was found in newborns with CDH compared to controls while DSPC kinetics was similar in both groups, which may contribute to severe respiratory failure in these patients.