Original Article

01/03/2005
A Review of Surfactant Kinetics in Newborn Respiratory Diseases

Virgilio P. Carnielli
Salesi Hosp, Ancona, Italy
University College, London, UK

Abstract

Deficiency or dysfunction of pulmonary surfactant plays a critical role in the pathogenesis of respiratory diseases in the newborn period. We describe the studies made by applying two recently developed methods to measure surfactant kinetics in humans. The first allows the measurement of endogenous surfactant phosphatidylcholine (PC) synthesis and kinetics by a constant intravenous infusion of glucose or fatty acids labeled with stable isotope 13C. The second method consists of endotracheal administration of a tracer dose of 13C-labeled dipalmitoil-phosphatiodylcholine (DPPC) to measure disaturatedphosphatidylcholine (DSPC) half-life and apparent pool size. We present the results of surfactant kinetic in some of the newborn respiratory diseases.

Background

Surfactant is a surface-active compound that provides surface-tension-lowering activity to the lungs.
Surfactant consist of about 90% lipids, of which the main component is phosphatidylcholine (PC) and about 10% surfactant specific proteins. Di-palmitoylphosphatidylcholine (DPPC) is about 60% of the PC species and it is the most important tensioactive component of surfactant. Deficiency or dysfunction of pulmonary surfactant plays a critical role in the pathogenesis of respiratory diseases in the newborn period.
The administration of exogenous surfactant in respiratory distress syndrome (RDS) has constituted one of the most important therapeutic advances in neonatology and it has rapidly become common practice for the treatment of RDS. This fact has also led to the increasing interest of studying surfactant characteristics not only in the immature lung but also in other newborn diseases such as pneumonia, bronchopulmonary dysplasia (BPD) and congenital diaphragmatic hernia (CDH). Futhermore it is of interest to study if the impact on surfactant kinetics of exogenous surfactant dosing, of different surfactant preparations and last but not least if different ventilatory modes or styles have or not a significant clinical impact on surfactant metabolism.
Surfactant kinetics has been measured in animal studies for decades by using radioactive isotopes. This approach however, is not suitable for research in humans and certainly not in infants. Therefore information on the surfactant system in humans ais scarce and mainly related to composition of surfactant samples obtained from tracheal aspirates and/or bronchoalveolar lavage (Hallmann 1991). We have recently developed methods based on safe stable isotopes that make surfactant kinetic studies ethically feasible in humans. The most obvious advantage of stable isotopes is that they are non-radioactive and they present no risk to the human subjects. Carbon 13 is a naturally occurring isotope present to the extent of approximately 1.11% of the carbon atoms. For the study of surfactant metabolism, substrates labelled with stable isotopes can be given intravenously or endotracheally. By infusing intravenously stable isotope labelled glucose and/or fatty acids the synthesis of surfactant (usually expressed as fractional synthesis rate=FSR) can be studied (Bunt 1998 , Caviccholi 2001). Labelled substrates are taken up by alveolar type II cells and used for the synthesis of surfactant components, which is secreted into the alveolar space and can be collected by sequential tracheal aspirates. PC or DSPC FSR, secretion time, peak time and half life can be calculated from the 13C enrichment decay curves over time. Instead we can trace pulmonary surfactant (Torresin 2000) by administering intratracheally stable isotope-labelled phospholipids, and the dilution of the tracer can give an estimate of surfactant pool size in the lung.
A summary of the key studies on surfactant kinetics by stable isotopes in the most important neonatal lung disease is reported below.

a) Newborns with RDS

- Intravenous administration of stable isotope PC or DSPC precursors

PC and DSPC secretion was measured in preterm infants (mean gestational age of 27 weeks) with severe RDS using intravascular infusions of glucose labelled with stable isotopes (Bunt 1998). The incorporation of the carbon 13 into surfactant PC palmitate began at about 20 hrs after the start of the isotope infusion, which most likely corresponds to the time required for palmitic acid synthesis from glucose, processing of surfactant PC in the alveolar type II cell, and secretion into the alveolar spaces. The PC palmitate enrichment was maximal at 70 hrs after the start of isotope infusion. Half life was found to be 113 hours.
This same stable isotope technology has also been used to demonstrate that antenatal glucocorticoids and postnatal surfactant treatments probably stimulate endogenous surfactant synthesis in ventilated infant s with RDS (Bunt 2000). In a group of preterm infants with a gestational age < 32 weeks and with RDS, the endogenous surfactant PC synthesis from glucose increased by 1.3 mg/kg/day per dose of exogenous surfactant. Although the increase of 1.3 mg/kg/day per dose surfactant is small compared with the surfactant pool sizes after treatment with exogenous surfactant, the importance of this finding is that endogenous surfactant synthesis is not suppressed by surfactant treatment and that there is no negative feedback mechanisms operating.
The use of exogenous surfactant administration is further supported by the finding of a low rate of PC synthesis from glucose (FSR = 2.7%/d) which could partially explain why preterm infants with RDS not receiving exogenous surfactant improve only after a few days (Bunt 1998). In fact the pool size of endogenous PC in preterm infants with RDS has been estimated to be very low and approximately 5 mg/kg (Hallman 1986 ).

- Intratracheal administration of stable isotope U 13C palmitic acid (PA) DPPC

We used the endotracheally stable isotope DPPC to trace exogenous surfactant and we reported for the first time on the direct measurement of the pharmacokinetics of exogenous surfactant DSPC in preterm infants with RDS. From the decay curve of the enrichment of U 13C palmitic acid (PA) DPPC in serial tracheal aspirates, we calculated the endogenous DSPC pool size to be 5.8 mg/kg in preterm with RDS before the first surfactant dose.
The mean DSPC half-life in this study was 34.2 hrs, which is very similar to data obtained through indirect methods in another group of preterm infants with RDS whose clinical characteristics were similar to those of the infants in our study (Hallman 1986). This result differs from the half life calculated by infusing U13C glucose intravenously, that resulted much longer (Bunt 1998). Among factors that could have caused longer surfactant half-lives in that study compared to this one are: the type of infusion (continuous versus bolus), the compartment used for administration (systemic circulation or trachea) and the difference between total surfactant pool and the alveolar pool. Half life reflects the DSPC disappearance rate from lung DSPC. Tracer disappearance can be caused either by an DSPC catabolism (loss of stable isotope DPPC molecules to the upper airways or to the blood-stream), or by a deacylation/reacylation pathway. In the latter case the tracer palmitic acid produced by breakdown of DSPC is reincorporated into newly synthesized non-DSPC molecules, thus it is lost from the DSPC pool.

b) Newborns with BPD

In a recent study (Cogo 2003) we compared surfactant kinetics in pre term infants with BPD and with no lung disease by endotracheal administration of U 13C palmitic acid (PA) DPPC. We found that DSPC half life in BPD newborns was 19.4 hrs which was significantly shorter than in age matched controls. In the BPD infants a shorter half life suggests a faster tracer breakdown. Elevated levels of pro-inflammatory cytokines (TNF-, IL 1, IL6 and IL8) in bronchoalveolar lavage have been found in term and pre term newborns BPD. We speculated that the inflammatory process leading to increased cytokines release could be responsible for the accelerated surfactant turnover and surfactant alterations, which in turns could exacerbate lung injury. In addition, we found despite a large apparent DSPC pool size, that epithelial lining fluid (ELF)-DSPC was lower in mechanically ventilated BPD newborns. The lower ELF DSPC could be explained by a reduced DSPC synthesis or secretion or by an increased DSPC catabolism. Unfortunately we did not measure in this group of patients de novo DSPC synthesis from the lung and we assumed ELF-DSPC as an indicator of the alveolar surfactant status. Seidner et al (1998) reported that in BPD baboons, despite an increased DSPC lung tissue pool, only 7% of the novo synthesized DSPC was secreted to the air spaces in 24 hrs and hypothesized an impaired DSPC secretion during BPD. Further studies in vivo in humans , tracing specific surfactant metabolic pathways simultaneously, are in progress to address this issue. We clearly demonstrated that the evolution of BPD is associated with alteration of DSPC kinetics and this is a new finding that can be used in future studies in which different ventilation styles, therapeutic interventions, and different levels of lung inflammation can be compared.

c) Newborns with pneumonia

To further explore the role of surfactant composition in lung inflammation we measured the amount of surfactant DSPC recovered from tracheal aspirates and its half life in newborns with pneumonia and compared them to normal lung. We found that term and preterm newborns with pneumonia exhibited much shorter DSPC half life than term newborns with normal lungs, suggesting a faster tracer breakdown. Furthermore in this study mean Oxygenation Index was negatively correlated with DSPC-half life (coefficient ? -0.41 p=0.03) suggesting that the degree of mechanical ventilation and oxygen exposure may act as promoter and propagator of injury especially in the lungs of preterm infants (Verlato 2003). Unfortunately in this study we did not measure de novo DSPC synthesis and/or pool size and we used the amount of DSPC recovered from tracheal aspirates as an indicator of the alveolar surfactant status. We found that DSPC amount in infants with lung disease was similar to that of term infants with normal lungs, possibly suggesting an adequate amount of exogenous surfactant delivered to our infants with lung disease.

d) Newborns with CDH

CDH remains an unsolved clinical problem, with high morbidity and mortality despite recent advances in respiratory support. It is still unclear whether a primary surfactant deficiency is present in human CDH. A recent study, in mechanically ventilated infants, reports similar concentrations of PC, phosphatidylglycerol and L/S ratios from bronchoalveolar lavage fluid of CDH and of age matched control infants, suggesting that primary surfactant deficiency is unlikely in infants with CDH (Ijsselstijn 1998). An even more recent study showed similar PC half life and pool size in CDH newborns who required ECMO support, compared with CDH newborns not treated with ECMO and with newborns with meconium aspiration syndrome (Janssen 2002).
We found moderately but significantly reduced amounts of DSPC and a marked reduction of SP-A from tracheal aspirates of CDH infants (Cogo 2003). Nonetheless by infusing stable isotopes intravenously we found comparable rates of endogenous DSPC synthesis in infants with CDH and in control neonates. Furthermore in a recent study we found that half life was significantly shorter in CDH infants compared with gestational age and postnatal age matched controls with normal lungs. Therefore we speculate that alteration of surfactant kinetics in CDH may be more related to enhanced catabolism than impaired synthesis. Larger studies will ascertain if alterations of surfactant DSPC in CDH infants are related to lung dysplasia or are secondary to mechanical ventilation lung injury.

e) Effect of Ventilation Mode on Surfactant Kinetics in Preterm Infants with RDS

Early HFOV in comparison with CV decreases exogenous surfactant requirements (Plavka 1999; Moriette 2001). This clinical finding can be explained by one, or by a combination of the following (a) enhanced synthesis (b) reduced catabolism (c) surfactant deficiency masked by the high mean airway pressure of HFOV. DSPC synthesis was found NOT to be different during HFOV and CV in Neonatal RDS using 13C Glucose as tracer (Merchack 2002). Hypotheses (b) and (c) remain untested. We used the “exogenous tracing method” (Torresin, AJRCCM 2000; 161:1584-89) and obtained preliminary data on surfactant DSPC half life (HL) in preterm infants with RDS, on HFOV or CV. Eighteen pre term infants all treated with exogenous surfactant soon after birth, were randomly assigned to elective HFOV or CV. DSPC Preliminary results indicate that surfactant DSPC half-life (HL) was significantly longer in the HFOV infants (59±25 h) in comparison with the CV group (40±13 h) p=0.03. From this preliminary work we collected evidence that HFOV likely spares surfactant in neonatal RDS.

f) Effect of Exogenous Surfactant Dosage on Surfactant Kinetics in Preterm Infants with RDS

Animal studies showed that the amount of exogenous surfactant does not affect surfactant HL, however no information is available in humans. Using the “exogenous tracing method” (Torresin, AJRCCM 2000; 161:1584-89) we performed studies with the objective of measuring surfactant kinetics in pre-term infants with RDS, assigned to receive doses of 100 mg/kg or 200 mg/kg of exogenous surfactant. Thirty-one preterm infants have been studies so far, 16 were treated with doses of 100 mg/kg (GR100) and 15 with doses of 200 mg/kg (GR200). All study patients were treated with porcine surfactant (Curosurf, Chiesi, ITALY) as rescue treatment for RDS. Birth weights were 972±109 g and 934±419 g (p=0.9) and gestational ages were 28±4 wks and 27±3 wks (p=0,2) in GR100 and GR200 respectively. In GR100, 10 infants (62.5%) received a second surfactant dose after 24±19 h and 3 (18.7%) a third dose; in GR200, 7 infants (46.7%) received a second dose after 31±17 h, none received 3 doses. Prenatal and neonatal clinical data, ventilator settings and gas exchange parameters were recorded daily. The two groups were similar with regards to all recorded clinical variables, including degree of respiratory disease and use of prenatal steroids. DSPC half life for the first doses were 23.7±9.8 vs 49.0±24.9 hours for GR100 and GR200 respectively (p=0.018). DSPC half life for the second doses were 33.9±7.9 vs 50.4±7.6 hours for GR100 and GR200 respectively (p=0.02). DSPC Half life for the third doses of the GR100 group was 45.0±8.6. The cumulative dose of exogenous surfactant was 172±72 and 273±88 mg/kg in the GR100 and GR200 groups respectively (0.001). In our series of patients by multiple regression analysis, HL was predicted only by study group (P=0.001) and not by BW, GA, number of surfactant doses or other clinical variables tested indicating that surfactant doses of 200 mg/kg given to pre-term infants for the treatment of RDS resulted in significantly longer DSPC half life compared to doses of 100 mg/kg.

In conclusion many efforts to explore surfactant kinetics in several lung diseases of newborn have been made and apparent surfactant pool size in RDS, BPD and CDH have been assessed. Surfactant synthesis in preterm RDS and half life in the above mentioned diseases and neonatal pneumonia have also been explored. The studies outline that quantitative and qualitative abnormalities of pulmonary surfactant contribute to the pathogenesis of several lung diseases in the newborn infant. Other studies by our group indicate also that variables such as ventilation modes or exogenous surfactant dosing do have a profound effect on surfactant kinetics. How these findings will influence future intervention strategies is not yet established but there is increasing evidence that surfactant metabolism, and the amount of surfactant present in the lungs of newborn infants plays an important role on lung function and lung pathophysiology.

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