Journal of Applied Physiology
Vol. 81, No. 6, pp. 2517-2522, December 1996
GAS EXCHANGE, MECHANICS, AND AIRWAYS

Surfactant inhibition by plasma: gestational age and surfactant treatment effects in preterm lambs

Machiko Ikegami, Celso M. Rebello, and Alan H. Jobe

Department of Pediatrics, Harbor-UCLA Medical Center, Torrance, California 90502

ABSTRACT

INTRODUCTION

METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

FOOTNOTES

REFERENCES

ABSTRACT

Ikegami, Machiko, Celso M. Rebello, and Alan H. Jobe. Surfactant inhibition by plasma: gestational age and surfactant treatment effects in preterm lambs. J. Appl. Physiol. 81(6): 2517-2522, 1996.The preterm infant with respiratory distress syndrome has edematous lungs and small amounts of surfactant that do not function normally. We reported that surfactant recovered from preterm lambs after surfactant treatment can have decreased sensitivity to inhibition of surface tension by plasma. We asked whether this augmented resistance to inhibition was dependent on lung development (gestational age) by testing sensitivity to plasma inhibition of 1) endogenous surfactant from preterm lambs and 2) surfactant from preterm lambs after treatment with an organic solvent-extracted natural sheep surfactant. Surfactant recovered after surfactant treatment of 121- or 128-days-gestation lambs had the same sensitivity to plasma inhibition as did the surfactant used to treat the lambs. Surfactant recovered from 134-days-gestation lambs had decreased sensitivity to inhibition. Lung maturation is a variable influencing surfactant inhibition by plasma.

respiratory distress syndrome; surface tension; phosphatidylcholine; lung development; Wilhelmy balance

INTRODUCTION

SURFACTANT METABOLISM AND FUNCTION in the alveoli are linked by interrelated pathways that involve changes in the structural forms, surfactant protein content, and biophysical properties of the alveolar surfactant (2, 30). This metabolism-function relationship is disrupted during lung injuries characterized by alveolar edema and inflammation (9, 21) and with preterm delivery and ventilation of the immature lung (20). The abnormalities that have been characterized in the preterm lung include decreased pool sizes of surfactant lipids (19), decreased amounts of the surfactant-specific proteins (26, 28, 30), increased minimum surface tensions (1), sequestration of surfactant within clots or hyaline membranes (7), and inhibition of surfactant function by soluble proteins in pulmonary edema fluid (12, 13).

Different surfactants also will have different characteristics. For example, relative to surfactant from the adult lung, surfactant from the preterm lung has a decreased density and an increased rate of conversion from surface-active to -inactive forms that correlated with a decreased surfactant protein-A (SP-A) content relative to phospholipids (28). Lipid-only surfactants are extremely sensitive to inhibition of surfactant function by soluble proteins, and the surfactant proteins SP-A, SP-B, and SP-C seem to act cooperatively to preserve the biophysical properties of surfactant (8, 10). The addition of SP-A to a surfactant that contains SP-B and SP-C can improve the function of the surfactant when administered with plasma to preterm rabbits (31). We recently reported (29) that surfactant recovered from lambs that received prenatal corticosteroid had improved function relative to surfactant from lambs not treated prenatally with corticosteroids. This result suggested that the maturational state of the lung might be a variable that would influence the sensitivity of surfactant to inactivation by plasma. Therefore, we hypothesized that lung maturation would influence the sensitivity of the endogenous surfactant pool to surfactant inhibition by plasma. We also have reported that surfactants used to treat the preterm lung (24) and the lungs of adult rabbits injured with N-nitroso-N-methylurethane have improved resistance to inactivation by plasma after recovery from the lungs (27). We also hypothesized that the ability of the preterm lung to confer resistance to plasma inhibition to a surfactant used for treatment will depend on the maturational state of the lung.

METHODS

Surfactant used for treatment. An organic solvent extract of sheep surfactant was used as the surfactant for those lambs treated with surfactant because it is similar to the natural-source surfactants used clinically (18). Sheep surfactant was isolated from alveolar lavage of adult sheep lungs by a three-step centrifugation procedure that includes a 0.8 M sucrose density gradient (13). The surfactant was mixed with chloroform-methanol (2:1) and mixed well with a magnetic stirrer. The solution was filtered through Whattman no. 1 filter paper to remove precipitated proteins. The residual surfactant lipids and proteins were extracted two more times with chloroform-methanol-saline (2:1:1). The extraction procedure removes SP-A but not SP-B or SP-C. The lipid extract of sheep surfactant was dried on a round bottom flask and suspended by using glass beads in 0.9% NaCl at a concentration of 25 mg/ml. This surfactant is highly surface active and effective for the treatment of surfactant deficiency in the preterm lambs (15).

Preterm lambs used for surfactant recovery. These experiments involve the use of surfactant from preterm lambs at different gestational ages for two protocols: the recovery of endogenous surfactant and the recovery of surfactant after surfactant treatment. For the studies using endogenous surfactant, preterm lambs were delivered at 128 days gestation (n = 9) and 134 days gestation (n = 5) and ventilated for 1 h to allow for secretion of the endogenous surfactant (17). We did not ventilate these premature lambs for longer periods to avoid death and severe lung injury (13). Surfactant was pooled from several 128-days-gestation lambs to yield sufficient material for testing. Lambs more immature than 128 days gestation were not used for studies of endogenous surfactant because the pool size of surfactant is too small (28). For studies of surfactant recovered after surfactant treatment, lambs were delivered at 121 days gestation (n = 4), 128 days gestation (n = 4), and 134 days gestation (n = 4); treated with surfactant; and ventilated for 3 h to permit the treatment surfactant to function within the alveolar environment (15).

Preterm lambs were delivered by cesarean section as previously described (15). Briefly, each pregnant ewe was sedated (1 g im ketamine) and given spinal-epidural anesthesia (10 ml 2% lidocaine-0.5% marcaine solution, 1:1, vol/vol). The anterior neck of the ewe was anesthetized (2% sq lidocaine), and an endotracheal tube was tied into the trachea followed by ventilation with a 10 ml/kg tidal volume by using a Harvard large-animal volume ventilator. The fetal head was exposed through midline abdominal and uterine incisions, and the fetus was anesthetized (10 mg/kg im ketamine and 0.1 mg/kg im acepromazine). The anterior fetal neck was anesthetized (2% sq lidocaine), and an endotracheal tube was tied into the trachea. Fetal lung fluid that could be easily aspirated by syringe was withdrawn, and the trachea tube was clamped. The umbilical cord was cut, and each lamb was weighed. The lambs to be treated with surfactant received 100 mg/kg of the lipid extract of sheep surfactant before the initiation of ventilation (15). The lambs were ventilated by using time-cycled, pressure-limited ventilators (Sechrist Industries, Anaheim, CA) with initial settings as follows: inspired O₂ fraction (FI_O2), 1.0; peak inspiratory pressure (PIP), 35 cmH₂O; positive end-expiratory pressure (PEEP), 3 cmH₂O; inspiratory time, 0.7 s; breathing rate, 40 breaths/min; and flow, 10 l/min. PIP was subsequently adjusted for each animal in an attempt to achieve arterial PCO₂ values of ~45 Torr, as assessed by frequent blood gas measurements. An attempt also was made to regulate PIP by adjusting tidal volumes to 7-10 ml/kg, as measured with a pneumotachometer (15). PIP values >35 cmH₂O for the lambs treated with surfactant and 40 cmH₂O for the lambs not treated with surfactant were not used to avoid pneumothorax. FI_O2 was adjusted for the surfactant-treated lambs to keep arterial PO₂ between 100 and 200 Torr. FI_O2 was kept at 1.0 for the lambs not treated with surfactant. The other ventilator settings were not changed.

A 5-Fr catheter was advanced into the aorta via an umbilical artery, and frequent blood samples were obtained for blood gas analysis. A constant infusion of 5% dextrose (4 ml ^· kg^{1 ·} h¹) was given, and blood pressure was recorded continuously. Rectal temperature was maintained at the normal body temperature for lambs of 38-39°C by using radiant heat and heating pads. The animals treated with surfactant received 10 µCi of ¹²⁵I-albumin at 2 h of age via the arterial catheter, and 1 h later a final blood sample was obtained for measurements of radiolabeled albumin and hemoglobin. Intravascular albumin was used as an indicator of transvascular pulmonary edema (20).

Alveolar lavage and surfactant isolation. At the end of the period of ventilation, the lambs were deeply anesthetized (50 mg/kg iv pentobarbital sodium) and exsanguinated. The lungs were removed from the thorax and filled by gravity with 0.9% NaCl at 4°C until fully distended. The alveolar lavage was recovered by using a syringe for premature lamb lung (20). The procedure was repeated five times, and the recovered alveolar lavages were pooled. The ewes that provided the lambs were ventilated for 1 h, killed with an overdose of pentobarbital sodium, and the lungs were removed from the chest. An alveolar wash was recovered, and surfactant was isolated from alveolar lavages by centrifugation (28). After an initial centrifugation at 140 g for 10 min, the supernatant was centrifuged at 40,000 g for 15 min. The pellet containing large-aggregate surfactant was resuspended in saline and centrifuged at 40,000 g for 15 min over 0.8 M sucrose. The interface was aspirated, diluted with saline, and centrifuged at 40,000 g for 15 min. The surfactant pellet then was resuspended in a small amount of saline for subsequent analysis.

Sensitivity of surfactant to plasma inhibition. Minimum surface tensions were measured by using a Wilhelmy balance with a platinum dipping plate after the fourth cycle from a maximum area of 64 cm² to a minimum area of 12.8 cm² at a temperature of 37°C (13). Area change was 3 min/cycle. Each surfactant was diluted in 35 ml saline to a final concentration of 0.05 mg lipid/ml. Plasma was then added and mixed with a glass rod by hand to achieve final concentrations of 0.4, 0.6, and 0.8 mg/ml protein, and surface tension measurements were repeated (6, 23). Surface area-surface tension loops were overlapping by the third or fourth cycle. Surface tension values at the fourth cycle are reported in RESULTS (27).

Material and analytical techniques. Lipids were extracted from surfactant samples with chloroform-methanol (2:1, vol/vol). After the aliquots of lipid extracts were dried, total lipids were determined by weight with a Cahn Electrobalance (Cahn-Ventron, Cerritos, CA). Saturated phosphatidylcholine (PC_sat) was isolated from lipid extracts by neutral alumina column chromatography after exposure to osmium tetroxide and quantified by phosphorus assay (3, 22). Monomer standard bovine serum albumin (Miles Laboratories, Elkhart, IN) was iodinated with chloramine T (20). After dialysis, over 97% of the radiolabel was protein bound, as indicated by trichloroacetic acid precipitation. SP-A was measured by radioimmunoassay (14).

Data analysis. All values are expressed as means ± SE. Differences between groups were tested by analysis of variance followed by the Student-Newman-Keuls multiple-comparison procedure.

RESULTS

Plasma inhibition of surfactant. Because the composition of plasma from preterm and adult sheep will have different compositions, we initially asked whether the source of the plasma would be a variable for these experiments. Surfactant recovered from lambs at 121, 128, and 134 days gestation that had been treated with surfactant was mixed with plasma from the lambs at the same gestation and compared with plasma from the ewes (Fig. 1). Inhibition of minimum surface tension by plasma from the ewes or the preterm lambs was similar for surfactant at each gestation. Therefore, aliquots of plasma from one adult sheep were frozen and used as the plasma source for all subsequent measurements.

Endogenous surfactant. The 128-days-gestation lambs were in severe respiratory failure with an average arterial PCO₂ of 87 Torr at 1 h of age, despite the use of 31 ± 2 cmH₂O ventilatory pressure (PIP  PEEP) (Table 1). The 134-days-gestation lambs were successfully ventilated on lower pressures. The amount of PC_sat and SP-A recovered in the large-aggregate surfactant was very low for the 128-days-gestation lambs. Both PC_sat and SP-A were increased at 134 days gestation. The pool sizes for these surfactant components increased further for surfactant from the ewes.

Table 1. Animals used to recover endogenous surfactant Age n Body Wt, kg Values at 1 h In Alveolar Lavage pH PaCO2, Torr PaO2/FIO2 , Torr PIP  PEEP, cmH2O PCsat, µmol/kg SP-A, mg/kg 128 Days gestation 9 2.7 ± 0.3  7.13 ± 0.06  87 ± 13  66 ± 12  31 ± 2  1.0 ± 0.2  0.08 ± 0.02  134 Days gestation 5 3.2 ± 0.1* 7.33 ± 0.01* 44 ± 1* 182 ± 42* 23 ± 1* 3.0 ± 0.7* 0.14 ± 0.01* Adult sheep 4 61.2 ± 2.4 11.3 ± 2.4 1.04 ± 0.07 Values are means ± SE; n = no. of lambs. PaCO2, arterial PCO2; PaO2, arterial PO2; FIO2, inspiratory O2 fraction; PIP, peak inspiratory pressure; PEEP, positive end-expiratory pressure; PCsat, saturated phosphatidylcholine; SP-A, surfactant protein-A. * P < 0.05 vs. 128-days-gestation lambs; P < 0.05 vs. 128- and 134-days gestation lambs.

The minimum surface tensions achieved on surface area compression for the endogenous surfactants from 128-days-gestation, 134-days-gestation, and adult sheep were similar and were <5 dyn/cm (Fig. 2). The addition of 0.4 mg plasma protein/ml had little effect on the minimum surface tension measurements. However, the surfactant from the 128-days-gestation lambs was the most sensitive to inhibition at higher plasma protein concentrations. The surfactant from the 134-days-gestation lambs was intermediate in sensitivity between the surfactant from the more immature lambs and the surfactant from adult sheep. Surfactant from adult sheep had a minimal surface tension <10 dyn/cm at a plasma concentration of 0.8 mg/ml.

Surfactant recovered after surfactant treatment. After 3-h ventilation, the lambs treated with surfactant had similar blood gas values and ventilatory requirements (Table 2). This result demonstrates the efficacy of surfactant even for very preterm lambs. There also was no difference in the amount of intravascular albumin that was recovered by alveolar lavage. Therefore, the amount of protein available to interfere with surfactant function was similar at the three gestational ages.

Table 2. Animals used to recover surfactant after surfactant treatment Age n Body Wt, kg Value at 3 h In Alveolar Lavage, %recovery of 125I-albumin pH PaCO2, Torr PaO2/FIO2 , Torr PIP  PEEP, cmH2O 121 Days gestation 4 1.9 ± 0.1  7.31 ± 0.01  41 ± 1  210 ± 48  19 ± 1  0.41 ± 0.10  128 Days gestation 4 2.7 ± 0.1* 7.32 ± 0.01  47 ± 2  209 ± 42  24 ± 2  0.47 ± 0.07  134 Days gestation 4 2.9 ± 0.1* 7.33 ± 0.01  45 ± 1  232 ± 23  21 ± 1  0.83 ± 0.22 Values are means ± SE; n = no. of lambs. * P < 0.05 vs. 121-days gestation lambs.

The organic solvent extract surfactant used to treat the preterm lambs had a low minimum surface tension in the absence of plasma (Fig. 3A). However, this surfactant was much more sensitive to inhibition by plasma than was the natural surfactant from adult sheep. The surfactants recovered from the preterm lambs at 121 or 128 days gestation, treated with surfactant, and ventilated for 3 h were similar in sensitivity to inhibition by plasma and not different from the lipid extract surfactant used to treat the lambs (Fig. 3B). However, surfactant from the 134-days-gestation lambs was more resistant to inhibition by plasma.

DISCUSSION

This study demonstrates that the endogenous surfactant recovered from the preterm lung is more sensitive to inhibition by plasma proteins than is surfactant from the adult. This sensitivity increases as gestation decreases. Similarly, surfactant used for treatment can become less sensitive to inhibition by plasma after exposure to the moderately preterm lung, an effect that is lost in the more preterm lung. These results add to the accumulating information indicating that surfactant deficiency of the preterm lambs with respiratory distress syndrome results from a number of factors. The surfactant pool size is low (19), and surfactant from preterm lambs is less effective as a treatment surfactant for surfactant deficiency (28). Several factors contribute to the abnormalities of surfactant from the preterm animals. SP-A content is decreased, and the rate of form conversion from large-aggregate active surfactant to small-aggregate inactive surfactant is increased (28). We now show increased sensitivity to inhibition by plasma proteins. Inhibition of surfactant in infants with respiratory distress syndrome is thought to contribute to deterioration in lung function and the need for subsequent doses of surfactant (11, 25). Airway samples from infants with respiratory distress syndrome have high minimum surface tensions, but the surfactant, when separated from soluble proteins, has good biophysical properties when tested in vitro (12). The infant with respiratory distress syndrome often has severe pulmonary edema as a component of the disease process (16, 20). An increased sensitivity of the surfactant of the preterm animal to plasma inhibition will compound the problem of surfactant inactivation. We did not find that plasma from preterm lambs or adult sheep had different abilities to inactivate surfactant.

Surfactants for clinical use from animal lung sources contain no SP-A, have different lipid compositions, and contain different amounts of SP-B and SP-C (8, 23). These surfactants were shown previously to be relatively sensitive to inhibition by plasma or plasma components (4, 6, 8). Animal-source surfactants with variable SP-B or SP-B and SP-C contents have different resistances to inactivation by proteins (8). We used an SP-A-deficient surfactant for these experiments to mimic the clinical situation. The preterm lambs treated with surfactant for these protocols did not have much pulmonary edema. The amount of labeled albumin given by intravascular injection that was recovered in the alveolar washes was low and comparable to measurements in term newborn lambs (20). We previously reported much higher recoveries of radiolabeled albumin in 121- and 128-days-gestation lambs (20). For present protocols, we kept tidal volumes at <10 ml/kg in an attempt to avoid volutrauma (5). We tried to avoid injury to the lungs to permit the comparison of surfactants recovered from lungs that had not been exposed to large amounts of protein-rich edema. We found that the more mature lung altered the surfactant used for treatment to make it more similar to the natural sheep surfactant. A likely explanation for this effect is the contribution of components from the preterm lung to the exogenously administered surfactant. We previously found SP-A in surfactant that did not initially contain SP-A after treatment of preterm lambs (15). Also, when 5% natural sheep surfactant is simply mixed with a bovine lung-derived surfactant that does not contain SP-A (Survanta, Ross Products, Columbus, OH), it makes that surfactant as resistant to inhibition by plasma as natural sheep surfactant (6). This effect of improved resistance to inhibition by plasma proteins was also found after surfactant treatment of rabbits with lungs injured with N-nitroso-N-methylurethane and after corticosteroid treatment of the fetal sheep (24, 27). Therefore, a general characteristic of the more mature lung is the ability to increase the resistance to plasma inhibition of surfactants used for treatment. This effect does not occur in the very preterm lung.

Another interaction of surfactant used for treatment with the preterm lung also was characterized by evaluating surfactant treatment responses of surfactant recovered from premature lamb lungs. Surfactants recovered after treatment of preterm lambs were more effective at improving compliances and lung volumes of surfactant-deficient preterm rabbits than were the surfactants used to treat the lambs (15). This phenomenon of surfactant "activation" was thought to occur by the association of endogenous surfactant components with the surfactant used for treatment. Therefore, the improved surfactant treatment responses and the enhanced resistance to inhibition by plasma may occur by similar mechanisms. Neither effect occurs in the very preterm lung (6).

This study describes two new mechanisms by which the very preterm infant have compromised surfactant function. The endogenous alveolar surfactant pool may have increased sensitivity to inhibition by plasma. Many plasma components as well as lipids, other proteins such as hemoglobin, and bilirubin also can inhibit surfactant function (10, 11). Sensitivity of surfactant from the preterm lambs to other inhibitors has not been evaluated, but it is also likely to be increased because the surfactant proteins also tend to protect from inactivation by other substances (10). Surfactants used for treatment of respiratory distress syndrome are relatively sensitive to inhibition by plasma, and the very preterm lung cannot augment resistance to inhibition. This result is consistent with the less consistent response to surfactant treatment and the need for repeated doses of surfactant for very preterm infants.

ACKNOWLEDGEMENTS

This research was supported by an National Institute of Child Health and Human Development Grant HD-12714 and by the Conselho Nacional de Desenvolvimento Cientifico Tecnologico Brazil Scholarship to Celso M. Rebello.

FOOTNOTES

Address for reprint requests: M. Ikegami, Dept. of Pediatrics, Harbor-UCLA Medical Center, 1124 W. Carson St., RB-1, Torrance, CA 90502.

Received 22 April 1996; accepted in final form 30 July 1996.

REFERENCES