Manuela Simonato

EIA and GC/MS analysis of 8-isoprostane in EBC of children with problematic asthma

Asthmatic airways are characterised by enhanced oxidative stress, which can be studied by measuring biomarkers, such as 8-isoprostane. The aims of the present study were: 1) to measure the concentrations of 8-isoprostane in exhaled breath condensate (EBC) and urine of children with problematic and well-controlled asthma; 2) to compare the concentrations of 8-isoprostane measured by gas chromatographic/negative ion chemical ionisation mass spectrometry (GC/NICI-MS) and by an enzymatic immunoassay (EIA). We recruited 20 asthmatic allergic children, 13 with well-controlled asthma and seven with problematic asthma. They underwent exhaled nitric oxide measurements and spirometry, and both EBC and urine samples were collected. 8-isoprostane was measured in EBC by GC/NICI-MS and EIA. 8-isoprostane concentrations in EBC were significantly higher in children with problematic asthma than in children with well-controlled asthma (p = 0.01). An acceptable reproducibility emerged between GC/NICI-MS and EIA (coefficient of reproducibility 11.5 pg·mL−1). 8-isoprostane levels measured in urine did not correlate with those measured in EBC. We showed that 8-isoprostane in EBC was significantly increased in children with problematic asthma, suggesting a role for oxidative stress in this asthma phenotype. In addition we found an acceptable reproducibility of EIA compared to GC/NICI-MS, even if the latter method had higher accuracy.

Dosing of Porcine Surfactant: Effect on Kinetics and Gas Exchange in Respiratory Distress Syndrome

OBJECTIVE: The goal was to study exogenous surfactant disaturated phosphatidylcholine (DSPC) kinetics in preterm infants with respiratory distress syndrome (RDS) who were treated with 100 or 200 mg/kg porcine surfactant. METHODS: Sixty-one preterm infants with RDS undergoing mechanical ventilation received, within 24 hours after birth, 100 mg/kg (N = 40) or 200 mg/kg (N = 21) porcine surfactant mixed with [U-13C]dipalmitoylphosphatidylcholine. Clinical and respiratory parameters were recorded, and DSPC half-life and pool size and endogenous DSPC synthesis rate were calculated. RESULTS: Clinical characteristics and short-term outcomes did not differ between groups. In the 100 mg/kg group, 28 infants (70%) received a second dose after 25 ± 11 hours and 9 (22.5%) a third dose after 41 ± 11 hours; in the 200 mg/kg group, 6 infants (28.6%) received a second dose after 33 ± 8 hours and 1 a third dose. The DSPC half-life was longer in the 200 mg/kg group (first dose: 32 ± 19 vs 15 ± 15 hours [P = .002]; second dose: 43 ± 32 vs 21 ± 13 hours [P = .025]). DSPC synthesis rates and pool sizes before the first and second doses did not differ between the groups. The 200 mg/kg group exhibited a greater reduction in the oxygenation index than did the 100 mg/kg group after the first (P = .009) and second (P = .018) doses. CONCLUSIONS: Porcine surfactant given to preterm infants with RDS at a dose of 200 mg/kg resulted in a longer DSPC half-life, fewer retreatments, and better oxygenation index values.

Disaturated-phosphatidylcholine and Surfactant protein-B turnover in human acute lung injury and in control patients

BackgroundPatients with Adult Respiratory Distress Syndrome (ARDS) and Acute Lung Injury (ALI) have low concentrations of disaturated-phosphatidylcholine and surfactant protein-B in bronchoalveolar lavage fluid. No information is available on their turnover.ObjectivesTo analyze disaturated-phosphatidylcholine and surfactant protein-B turnover in patients with ARDS/ALI and in human adults with normal lungs (controls).Methods2H2O as precursor of disaturated-phosphatidylcholine-palmitate and 113C-Leucine as precursor of surfactant protein-B were administered intravenously to 12 patients with ARDS/ALI and to 8 controls. Disaturated-phosphatidylcholine and surfactant protein-B were isolated from serial tracheal aspirates, and their fractional synthetic rate was derived from the 2H and 13C enrichment curves, obtained by gas chromatography mass spectrometry. Disaturated-phosphatidylcholine, surfactant protein-B, and protein concentrations in tracheal aspirates were also measured.Results1) Surfactant protein-B turned over at faster rate than disaturated-phosphatidylcholine both in ARDS/ALI patients and in controls. 2) In patients with ARDS/ALI the fractional synthesis rate of disaturated-phosphatidylcholine was 3.1 times higher than in controls (p < 0.01), while the fractional synthesis rate of surfactant protein-B was not different. 3) In ARDS/ALI patients the concentrations of disaturated-phosphatidylcholine and surfactant protein-B in tracheal aspirates were markedly and significantly reduced (17% and 40% of the control values respectively).Conclusions1) Disaturated-phosphatidylcholine and surfactant protein-B have a different turnover both in healthy and diseased lungs. 2) In ARDS/ALI the synthesis of these two surfactant components may be differently regulated.

Simplified method for microlitre deuterium measurements in water and urine by gas chromatography–high‐temperature conversion–isotope ratio mass spectrometry

Deuterium (2H) in water and urine can be measured by off-line and, more recently, on-line techniques using isotope ratio mass spectrometry (IRMS). We describe a new simple on-line pyrolysis method for the analysis of 2H/1H in water and urine samples by continuous flow IRMS, normally used for 2H/1H measurements in organic compounds. A deactivated column connected the split injector to a high-temperature conversion reactor (TC HD), and 0.5 microL of sample was injected. Accuracy and precision were determined with Vienna Standard Mean Ocean Water (VSMOW), Standard Light Antarctic Precipitation (SLAP), and Greenland Ice Sheet Precipitation (GISP). The range of linearity was measured with a calibration curve of enriched water from 0 up to 0.1 atom percent excess (APE) (i.e. -72 up to 6323 delta per mil (deltaD per thousand)) with a precision of <5 per thousand and accuracy ranging between 1 and 55 per thousand. Blinded reanalysis of urine samples by an equilibration device (Gas Bench) and by a dedicated pyrolysis system (TC/EA) was performed and results compared by the Bland-Altman test. Enrichments ranged between 600 and 2400 per thousand deltaD(VSMOW) with a precision of +/-5 per thousand. Urine enrichments described by our method were strongly correlated with values obtained by Gas Bench and TC/EA (p < 0.0001). There was a significant memory effect that was reduced by injecting the sample 15 times and discarding the first 10 injections, together with accurate furnace conditioning and appropriate cleaning of the syringe. Data indicate that the method is accurate, and that it can be used for water and urine deuterium determination when a Gas Bench or TC/EA instrument is not available and the amount of sample is limited.

Dexamethasone therapy in preterm infants developing bronchopulmonary dysplasia: effect on pulmonary surfactant disaturated-phosphatidylcholine kinetics.

The role of corticosteroid in severe bronchopulmonary dyplasia (BPD) is still debated. Scanty data are available on the corticosteroids effect on surfactant metabolism. Our objective was to compare surfactant kinetics in preterm infants with developing BPD, before and after dexamethasone (DEXA) treatment. Twenty-eight studies were performed in 14 preterm infants (birth weight 786 ± 192 g, gestational age 26 ± 1 wk) on high ventilatory setting, before (age 22 ± 11 d) and after (age 33 ± 11 d) DEXA. 13C-labeled dipalmitoyl-phosphatidylcholine (DPPC) was administered endotrachelly to trace pulmonary surfactant. Surfactant disaturated-phosphatidylcholine (DSPC) kinetics and pools were calculated from DSPC 13C-enrichment curves of serial tracheal aspirates and bi-compartmental analysis. Total protein and myeloperoxidase (MPO) activity in tracheal aspirates were also measured and expressed per ml of Epithelial Lining Fluid (ELF). After DEXA, DSPC alveolar pool increased significantly from 8.2 ± 7.6 to 10.6 ± 11.3 mg/kg (p = 0.039), total proteins and MPO were reduced from 8.8 ± 8.6 to 3.1 ± 2.1 mg/ml ELF (p = 0.046) and from 1822 ± 1224 to 1261 ± 987 mU/mlELF (p = 0.028) respectively. In conclusion, DEXA treatment in mechanically ventilated preterm infants with severe respiratory failure and at high risk of developing BPD, significantly reduced inflammatory markers and increased alveolar surfactant DSPC pool.

Metabolic precursors of surfactant disaturated-phosphatidylcholine in preterms with respiratory distress

Our objective was to study the metabolic precursors of surfactant disaturated-phosphatidylcholine (DSPC) in preterm infants with respiratory distress syndrome (RDS) on mechanical ventilation. We performed 46 DSPC kinetic studies in 23 preterms on fat-free parenteral nutrition and mechanical ventilation (birth weight = 1167 ± 451 g, gestational age = 28.5 ± 2.0 weeks). Eight infants received a simultaneous intravenous infusion of U13C-glucose and [16,16,16]2H-palmitate, eight infants received U13C-glucose and 2H2O, and seven received U13C-palmitate and 2H2O. Surfactant DSPC kinetics were calculated from the isotopic enrichments of DSPC-palmitate from sequential tracheal aspirates and its metabolic precursors in plasma or urine. DSPC fractional synthesis rate (FSR) was 17 ± 11, 21 ± 16, and 15 ± 6%/day from glucose, palmitate, and body water, respectively (P = 0.36). DSPC-FSR from U13C-glucose and 2H2O were significantly correlated and yielded similar estimates (difference of –0.1 ± 3%) (P = 0.91). The difference in the 15 infants receiving palmitate versus 2H2O or palmitate versus glucose was +6.0 ± 12%/day (P = 0.21). There was a significant correlation between DSPC-FSRs from plasma glucose and plasma FFA. The contribution of glucose versus palmitate to DSPC-FSR was 49 ± 20% versus 51 ± 20%, respectively. Plasma glucose and FFA showed similar contributions to DSPC-FSR in infants with RDS and fat-free parenteral nutrition. FSRs from 2H2O or glucose were highly correlated.

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. 2H2O as a precursor of DSPC and 1-13C-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 2H and 13C 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.

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.

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

Background Bacterial 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 surfactants major component, disaturated-phosphatidylcholine (DSPC), in neonatal pneumonia, and we compared our findings with those obtained from control newborn lungs. Methods We 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. Results The 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). Conclusions We 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.

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

Background It 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. Methods Twenty-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. Results DSPC 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. Conclusions WBH does not alter DSPC HL and PS in newborn infants with no clinical apparent lung disease.