Alveolar Lavage and Lavaged Lung Tissue Phosphatidylcholine Composition During Fetal Rabbit Development SEAMUS A. ROONEY and LAURICE I. GOBRAN, Department of Pediatrics, Yale University School of Medicine, New Haven, CT 06510
the respiratory distress syndrome of the newborn, the major cause of morbidity and mortality among premature infants in developed countries (3,4). The fetal lung begins to produce appreciable amounts of surfactant towards the end of gestation (2,3), and it has been shown that corticosteroids (3) and thyroxine (5) can accelerate fetal lung maturation and surfactant production. We have previously measured the phospholipid content and composition of lung lavage during the latter part of gestation in fetal rabbits and shown that there is a fourfold increase in the amount of total phospholipid between 27 and 31 (full term) days' gestation (6). During the same period, phosphatidylcholine increased from 29% of the total phospholipid to 80% (6). We have also shown that cortisol (7) and thyroxine (8) increase the amount of phosphatidylcholine in fetal rabbit lung lavage. However, there is little information on the nature of the molecular species of phosphatidylcholine in lung lavage during fetal development although detailed studies have been carried out on intact lung tissue (9). The purpose of the present study was to examine changes in the amount of saturated phosphatidylcholine and in the fatty acid composition of phosphatidylcholine from lung lavage and lavaged lung tissue during develop-' ment of the fetal rabbit. Such basic information is essential in elucidating the mechanism of surfactant production and the effects of hormones thereon during fetal development.
ABSTRACT
P u l m o n a r y surfactant, the major surface-active component of which in the adult is dipalmitoylglycerophosphocholine, can be obtained by lavaging lungs with physiological saline. We have previously shown that there is an increase in the amount of phosphatidylcholine in fetal rabbit lung lavage during the latter part of gestation. We have now measured the amount of disaturated phosphatidylcholine as well as the fatty acid composition of phosphatidylcholine in lung lavage from fetal rabbits during the period 27 days' gestation to full term (31 days). There was no developmental change in the amount of disaturated phosphatidylcholine during the period examined. About 50% of the total phosphatidylcholine was disaturated which is approximately the same as in adult rabbit lung lavage. The fatty acid composition, however, did change. There was an increase in the amount of 16:0 from about 20% of the total fatty acids in phosphatidylcholine at 27-28 days to about 60% at full term, after birth, and in the adults. There was a corresponding decrease in the amounts of 14:0, 18:0, and longer chain fatty acids, most of which were saturated. In the lavaged lung tissue, there was a 2.6-fold increase in the percentage of phosphatidylcholine which was disaturated during the period 27-31 days' gestation. It had not decrease to the adult value 24 hr after birth.
MATERIALS AND METHODS
INTRODUCTION
The lung is lined with surface-active material, pulmonary surfactant, which is believed to lower surface tension at the air-liquid interface and prevent alveolar collapse (1). Surfactant can be obtained from the lung by lavaging with saline. It has been shown to be largely phospholipid of which phosphatidylcholine is the most abundant component (1,2). Dipalmitoylglycerophosphocholine is believed to be the major surface-active component of pulmonary surfactant (1,2). Insufficient surfactant at birth can lead to
T i m e d p r e g n a n t rabbits (New Zealand W h i t e ) were p u r c h a s e d from Glochester Rabbitry, Chepachet, RI. The doe was sacrificed with intravenous sodium pentobarbital (80 mg/kg). The fetuses were immediately delivered by cesarean section and sacrificed with intraperitoneal sodium pentobarbital (30 rag) prior to breathing, while still in their amniotic sacs. Newborn animals were similarly sacrificed 1-24 hr after delivery by cesarean section, during which time they were kept in an incubator at constant temperature (31 C) and humidity. The lungs were lavaged in situ, via a tracheal cannula, with five 1-3 ml aliquots of 0.9%
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TABLE I Disaturated Phosphatidylcholine Content of Lung Lavage and Lavaged Lung Tissue from Fetal, Newborn, and Adult Rabbits a Lung lavage Lung tissue Disaturated phosphatidylcholine as % of total phosphatidylcholine Fetuses
Newborns
Gestational age (days) 27 28 29 30 31 Period after birth (hours)
49.3±1.9 55.5±0.9 46.0±5.3 50.2±4.4 52.8±3.0
27.0±3.2 34.0±4.5 34.1±3.8 35.2±3.3 41.6±2.6
54.0 ± 1.4
42.2 47.3 57.6 45.8 45.2 28.6
l
3 6 18 24 Adult b
± 1.8 ± 1.5 ± 0.8 ± 3.9 ± 1.7 + 1.3
aThe data are the means (±SE) from 3-6 experiments in each of which lavage and tissue from all fetuses in 1-2 litters were combined. The newborn and adult data are from 3-5 animals. bThe adult data are from a previous study (14). NaC1. The lavage liquid was c e n t i f u g e d at 1000 x g to r e m o v e cellular material. Lipids were ext r a c t e d f r o m the l y o p h i l i z e d lavage a n d tissue as described previously (6,10). P h o s p h a t i d y l c h o l i n e was isolated b y t h i n layer c h r o m a t o g r a p h y (TLC) on Q u a n t u m LQD plates ( Q u a n t u m Industries, Fairfield, N J) in chloroform-methanol-7 M NH4OH (60:35:4, b y vol). It was eluted f r o m t h e gel a n d quantirated b y p h o s p h o r u s assay as described previously (10). P h o s p h a t i d y l c h o l i n e was saponified w i t h m e t h a n o l i c KOH (11). T h e f a t t y acids were m e t h y l a t e d , a n d t h e m e t h y l esters were analyzed b y gas liquid c h r o m a t o g r a p h y as described previously (12). D i s a t u r a t e d p h o s p h a t i d y l c h o l i n e was separated from total phosphatidylcholine from lung tissue f o l l o w i n g o s m i u m t e t r o x i d e t r e a t m e n t b y the m e t h o d of Mason et al. (13) as d e s c r i b e d previously (14). Lavage d i s a t u r a t e d p h o s p h a t i d y l c h o l i n e was similarly m e a s u r e d e x c e p t t h a t it was s e p a r a t e d f r o m t h e oxidized p h o s p h a t i d y l c h o l i n e b y TLC o n Q u a n t u m LQD plates in chloroform-methanol-7 M NH4OH ( 7 0 : 3 0 : 3 . 5 , b y vol) (13). Q u a n t i t a t i o n was b y the u l t r a m i c r o p h o s p h o r u s assay d e s c r i b e d b y B a r t l e t t (15). In this p r o c e d u r e , 86% o f standard dipalmitoylglycerophosphocholine and 1.5% of s t a n d a r d d i o l e o y l g l y c e r o p h o s p h o c h o line were r e c o v e r e d in t h e p h o s p h a t i d y l c h o l i n e band. Standard p h o s p h o l i p i d s were p u r c h a s e d
f r o m Serdary, L o n d o n , O n t a r i o , C a n a d a a n d s t a n d a r d f a t t y acid m e t h y l esters f r o m Supelco, Bellefonte, PA. O t h e r chemicals were r e a g e n t grade or better. R ESU LTS
In t h e lung lavage f r o m fetal rabbits, a b o u t half of t h e t o t a l p h o s p h a t i d y l c h o l i n e was disaturated throughout the developmental period e x a m i n e d ( T a b l e I). A similar a m o u n t of phosp h a t i d y l c h o l i n e is d i s a t u r a t e d in lung lavage f r o m t h e adult rabbit. T h e f a t t y acid c o m p o s i t i o n o f p h o s p h a t i d y l c h o l i n e from fetal a n d n e w b o r n r a b b i t lung lavage is s h o w n in T a b l e II. In a g r e e m e n t w i t h t h e finding w i t h respect to d i s a t u r a t e d p h o s p h a t i d y l c h o l i n e , t h e r e was little change in t h e t o t a l a m o u n t o f s a t u r a t e d f a t t y acids d u r i n g developm e n t . T h e c o m p o s i t i o n of t h e f a t t y acids, h o w ever, did change. T h e r e was an increase in t h e a m o u n t of 16:0 a n d a c o r r e s p o n d i n g decrease in t h e a m o u n t s o f 14:0, 18:0, a n d longer c h a i n ( > 18 c a r b o n a t o m s ) f a t t y acids f r o m 27 days' g e s t a t i o n to full term. T h e f a t t y acid c o m p o s i t i o n of lung lavage p h o s p h a t i d y l c h o l i n e at 31 days' g e s t a t i o n a n d 6 hr a f t e r b i r t h was similar to t h a t of adults (12). A t 31 days' g e s t a t i o n a n d a f t e r b i r t h , as well as in adults (12), 16:0 acc o u n t e d for a b o u t 60% of t h e t o t a l a n d 90% of t h e s a t u r a t e d f a t t y acids. Thus, t h e b u l k o f d i s a t u r a t e d p h o s p h a t i d y l c h o l i n e in l u n g lavage at this d e v e l o p m e n t a l stage m u s t b e dipalmiLIPIDS, VOL. 12, NO. 12
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S.A. R O O N E Y A N D L.I. G O B R A N
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L I P I D S , V O L . 12, N O . 12
FETAL LUNG PHOSPHATIDYLCHOLINE
1053
TABLE III Fatty Acid Composition of Phosphatidylcho]ine from Lavaged Lung Tissue of Fetal Rabbits a Fatty acid methyl ester n 14:0 14:2 15:0 16:0 16:1 18:0 18:1 18:2 18:3 22:0 20:4 Others
24 b
Gestational age (days) 28 29
27
30
31
4
3
3
3
3
3
3.3• 1.7• trace 35.9• 7.2• 6.8• 24.9• 1.2 13.9• trace trace 3.4• 1.0 2.9• 1.4
0.8• trace trace 39.2• 4.4• 7.5• 23.8• 14.6• 1.2 1.2• 0.7• 6.1• 1.7•
1.6e0.1 1.7• trace 47.0• 5.2• 5.8 • 17.5• 11.5• 0.9• 0.8• 6.8• 1.2•
2.5• 0.9• 0.6• 49.0• 6.5• 5.6• 15.6• 11.3• 1.1 0.5 • 0.6• 5.7• 1.0•
2.3• 0.6• 0.5• 49.1• 7.2• 4.7• 15.0• 11.6• 0.9• 0.7• 6.5• 0.8•
3.2• trace 0.8• 51.5• 8.4• 4.7• 13.1 • 12.2• 0.8• 0.6• 3.6• 1.4 1.1 •
aTrace, < 0.5%. Other details as in Table II. bThe lungs from the 24 day fetal rabbits were not lavaged prior to lipid extraction. t o y l g l y c e r o p h o s p h o c h o l i n e . (The fatty acid c o m p o s i t i o n of the disaturated species was n o t determined because of the small a m o u n t s of material available for analysis). At 29 and 30 days' gestation, 16:0 a c c o u n t e d for about 50% of the total and 76-77% of the saturated fatty acids so that, even at this stage of d e v e l o p m e n t , d i p a l m i t o y l g l y c e r o p h o s p h o c h o l i n e w o u l d be the m a j o r disturated species. At 27 and 28 days, however, 16:0 a c c o u n t e d for only a b o u t 20% of the total and 30% of the saturated f a t t y acids. Thus, at this d e v e l o p m e n t a l stage, dipalm i t o y l g l y c e r o p h o s p h o c h o l i n e is p r o b a b l y a m i n o r c o m p o n e n t of lung lavage phosphatidylcholine. Long chain fatty acids, largely saturated, a c c o u n t e d for 31% of the total at 27 days' gestation and 12% at 28 days b u t only 2-3% at 29-31 days. Arachidonic acid (20:4), w h i c h is c h a r a c t e r i s t i c a l l y absent from surfactant, accounted for 5% of the total at 27 days. 0.5-1% at 28-30 days, and was not seen at 31 days or after birth. There was an increase in the a m o u n t of 16:1 from 4% of the t o t a l at 27 days to 12% at 28-29 days and to a b o u t 16% at full term. In the lavaged lung tissue, disaturated phosphatidylcholine increase from 27% of the t o t a l phosphatidylcholine at 27 days' gestation to 42% at 31 days (Table I). It increased even further after birth and did not decrease to the adult value during the period examined. The fatty acid c o m p o s i t i o n of p h o s p h a t i d y l c h o l i n e from lavaged lung tissue is shown in Table III. There was little change during the period 24-31 days' gestation other than an increase in the a m o u n t of 16:0 from 36% of the total fatty acids to 52% and a corresponding decrease in the a m o u n t of 18:1 from 25% to 13%. These changes in fatty acid c o m p o s i t i o n are consistent
with the d e v e l o p m e n t a l increase in disaturated phosphatidylcholine. By 31 days' gestation, the fatty acid c o m p o s i t i o n of lung tissue phosphatidylcholine was similar to that of adults (14). DISCUSSION
Surfactant appears to be synthesized by the fetal lung towards the end of gestation (2) and largely stored, presumably in lamellar inclusion bodies in type II alveolar epithelial cells (16), until after birth when it is released in large q u a n t i t y into the alveoli (6,17). It has been shown that there is an increase in the a m o u n t of phospholipid, particularly phosphatidylcholine, in lung lavage during the latter part of gestation in the fetal rabbit (6) and lamb (18). However, the total a m o u n t in the lavage even at full term accounts for no m o r e than 1% of the total p u l m o n a r y phospholipid or phosphatidylcholine (8). One day after birth, on the other hand, 7% of total p u l m o n a r y phospholipid and 10% of total p u l m o n a r y p h o s p h a t i d y l c h o l i n e is recovered in the lavage (8). There are also changes in the c o m p o s i t i o n of lung lavage phospholipid during development. At 27 days' gestation in the rabbit, for instance, phosphatidylcholine accounts for only 29% of the total phospholipid while sphingomyelin accounts for 38% (6). After birth (6) and in the adult (10), phosphatidylcholine accounts for at least 80% of the total but sphingomyelin accounts for less than 3%. Similarly, as shown in this paper, there are changes in the fatty acid c o m p o s i t i o n of p h o s p h a t i d y l c h o l i n e from lung lavage during fetal development. F r o m these studies, it is apparent that dipalmitoylglycerophosphocholine, the major surface-active comp o n e n t of p u l m o n a r y surfactant in adults (1,2), LIPIDS, VOL. 12, NO. 12
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S.A. ROONEY AND L.I. GOBRAN
is only a relatively m i n o r c o m p o n e n t o f lung lavage p h o s p h o l i p i d b e f o r e a b o u t 29 d a y s ' gest a t i o n in the rabbit. It is o f i n t e r e s t t h a t the surface activity o f lung lavage p h o s p h a t i d y l c h o line also begins t o increase at t h e same gestational age (19). There are few previous studies on the f a t t y acid c o m p o s i t i o n o f lung lavage p h o s p h a t i d y l choline during fetal d e v e l o p m e n t . Fijiwara et al. (18) r e p o r t e d t h a t t h e a m o u n t o f 16:0 in p h o s p h a t i d y l c h o l i n e f r o m fetal lamb lung lavage increased w i t h increasing gestational age. F r o m f a t t y acid analysis, these w o r k e r s calculated t h a t the p e r c e n t a g e o f m i n i m u m d i s a t u r a t e d p h o s p h a t i d y l c h o l i n e increased during t h e same period (18). Gluck et al. (20) also r e p o r t e d t h a t t h e r e was m o r e 16:0 in p h o s p h a t i d y l c h o l i n e f r o m lung lavage o f fetal rabbits at 31 days' g e s t a t i o n t h a n in t h a t f r o m t h o s e at 23 days. We have carried o u t studies o n tracheal and gastric liquids f r o m n e w b o r n h u m a n s at b i r t h (sources o f alveolar liquids) and f o u n d t h a t p h o s p h a t i d y l c h o l i n e in the materials from p r e m a t u r e i n f a n t s c o n t a i n e d less 16:0 and m o r e 18 c a r b o n f a t t y acids t h a n in t h a t f r o m full t e r m i n f a n t s (21). Similar data has b e e n rep o r t e d for a m n i o t i c fluid ( a n o t h e r source o f fetal s u r f a c t a n t ) f r o m h u m a n s (22-25) and sheep (26). A n increase in t h e a m o u n t o f p u l m o n a r y d i s a t u r a t e d p h o s p h a t i d y l c h o l i n e during fetal d e v e l o p m e n t has also b e e n r e p o r t e d b y others. Fujiwara et al. (18) and B r u m l e y et al. (27) reported a d e v e l o p m e n t a l increase in the a m o u n t of d i s a t u r a t e d p h o s p h a t i d y l c h o l i n e in lavaged and i n t a c t fetal lamb lung, respectively. S o o d s m a et al. (9) r e c e n t l y r e p o r t e d increases in the a m o u n t s o f d i s a t u r a t e d p h o s p h a t i d y l c h o line and d i p a l m i t o y l g l y c e r o p h o s p h o c h o l i n e in i n t a c t lungs f r o m fetal rabbits during t h e p e r i o d 23.5 to 30.5 d a y s ' gestation. The increased a m o u n t o f d i s a t u r a t e d phosp h a t i d y l c h o l i n e in lavaged lung tissue w i t h increasing g e s t a t i o n a l age m a y reflect increased synthesis and storage o f s u r f a c t a n t prior to birth. It is o f i n t e r e s t t h a t the a m o u n t o f disaturated p h o s p h a t i d y l c h o l i n e in t h e lavaged tissue does n o t decrease i m m e d i a t e l y after b i r t h w h e n t h e r e is a t r e m e n d o u s increase in t h e a m o u n t o f p h o s p h a t i d y l c h o l i n e - a b o u t h a l f o f w h i c h is d i s a t u r a t e d - in t h e lavage (6,17). It is possible t h a t n o t all o f t h e s t o r e d s u r f a c t a n t is released i n t o the alveoli in the first 24 hr after birth. The l o w e r level o f d i s a t u r a t e d p h o s p h a t i d y l c h o line f o u n d in t h e adult lung tissue m a y n o t be r e a c h e d for s o m e t i m e a f t e r birth. ACKNOWLEDGMENT This work was supported by a grant (HD-10192) LIPIDS, VOL. 12, NO. 12
from the National Institute of Child Health and Human Development, USPHS. REFERENCES 1. Goerke, J. Biochim. Biophys. Acta 344:241 (1974). 2. Van Golde, L.M.G., Am. Rev. Respir. Dis. 114:977 (1976). 3. Farrell, P.M., and M.E. Avery, Am. Rev. Respir. Dis. 111:657 (1975). 4. Farrell, P.M., and R.E. Wood, Pediatrics 58:167 (1976). 5. Wu, B., Y. Kikkawa, M.M. Orzalesi, E.K. Motoyama, M. Kaibara, C.J. Zigas, and C.D. Cook, Biol. Neonat 22:161 (1973). 6. Rooney, S.A., T.S. Wai-Lee, L. Gobran, and E.K. Motoyama, Biochim. Biophys. Acta 431:447 (1976). 7. Rooney, S.A., L. Gobran, I. Gross, T.S. Wai-Lee, L.L. Nardone, and E.K. Motoyama, Biochim. Biophys. Acta 450:121 (1976). 8. Rooney, S.A., and E.K. Motoyama, in "Pulmonary Macrophage and Epithelial Cells," 16th Annual Hanford Biology Symposium, Energy Research and Development Administration Conference 760927, 1977, p. 162. 9. Soodsma, J.F., L.C. Mims, and R.D. Harlow, Biochim. Biophys. Acta 424:159 (1976). 10. Rooney, S,A., P.M. Canavan, and E.K. Motoyama, Biochim. Biophys. Acta 360:56 (1974). 11. Rooney, S.A., and H. Goldfine, J. Bacteriol. 111:531 (1972). 12. Rooney, S.A., B.A. Page-Roberts, and E.K. Motoyama, J. Lipid Res. 16:418 (1975). 13. Mason, R.J., J. Nellengogen, and J.A. Clements, J. Lipid Res. 17:281 (1976). 14. Rooney, S.A., L.L. Nardone, D.L. Shipiro, E.K. Motoyama, L. Gohran, and N. Zaehringer, Lipids 12:438 (1977). 15. Bartlett. G.R., J. Biol. Chem. 234:466 (1959). 16. Rooney, S.A., in "Lung Cells in Disease," Edited by A. Bouhuys, North Holland, Amsterdam, The Netherlands, 1976, p. 147. 17. Rooney, S.A., L.I. Gobran, and T.S. Wai-Lee, J. Clin. Invest. 60:754 (1977). 18. Fujiwara, T., F.H. Adams, S. Sipos, and A. EI-Salawy, Am. J. Physiol. 215:375 (1968). 19. Gluck, L., E.K. Motoyama, H.L. Smits, and M.V. Kulovich, Pediatr. Res. 1:237 (1967). 20. Gluck, L., R.A. Landowne, and M.V. Kulovich, Pediatr. Res. 4:352 (1970). 21. Motoyama, E.K., Y, Namba, and S.A. Rooney, Clin. Chim. Acta 70:449 (1976), 22. Ekelund, L., A. Gosta, and B. Astedt, J. Obstet. Gynaecol. Br. Commonw. 80:912 (1973). 23. Roux, J.F., J. Nakamura, and M. Frosolono, Am. J. Obstet. Glynecol. 119:838 (1974). 24. Frantz, T., T. Lindback, J. Skjaeraasen, and S. Graven, Acta Obstet. Gynecol. Scand. 54:33 (1975). 25, Schirar, A., J.P. Vielh, L.G. Alcindor, and J.P. Gautray, Am. J. Obstet. Gynecol. 121:653 (1975). 26, Ogawa, Y., Tohoku J. Exp. Med. 108:307 (1972). 27. Brumley, G.W., V. Chernick, W.A. Hodson, C. Normand, A. Fenner, and M.E. Avery, J. Clin. Invest. 46:863 (1967).
[Received July 18, 1977]
the respiratory distress syndrome of the newborn, the major cause of morbidity and mortality among premature infants in developed countries (3,4). The fetal lung begins to produce appreciable amounts of surfactant towards the end of gestation (2,3), and it has been shown that corticosteroids (3) and thyroxine (5) can accelerate fetal lung maturation and surfactant production. We have previously measured the phospholipid content and composition of lung lavage during the latter part of gestation in fetal rabbits and shown that there is a fourfold increase in the amount of total phospholipid between 27 and 31 (full term) days' gestation (6). During the same period, phosphatidylcholine increased from 29% of the total phospholipid to 80% (6). We have also shown that cortisol (7) and thyroxine (8) increase the amount of phosphatidylcholine in fetal rabbit lung lavage. However, there is little information on the nature of the molecular species of phosphatidylcholine in lung lavage during fetal development although detailed studies have been carried out on intact lung tissue (9). The purpose of the present study was to examine changes in the amount of saturated phosphatidylcholine and in the fatty acid composition of phosphatidylcholine from lung lavage and lavaged lung tissue during develop-' ment of the fetal rabbit. Such basic information is essential in elucidating the mechanism of surfactant production and the effects of hormones thereon during fetal development.
ABSTRACT
P u l m o n a r y surfactant, the major surface-active component of which in the adult is dipalmitoylglycerophosphocholine, can be obtained by lavaging lungs with physiological saline. We have previously shown that there is an increase in the amount of phosphatidylcholine in fetal rabbit lung lavage during the latter part of gestation. We have now measured the amount of disaturated phosphatidylcholine as well as the fatty acid composition of phosphatidylcholine in lung lavage from fetal rabbits during the period 27 days' gestation to full term (31 days). There was no developmental change in the amount of disaturated phosphatidylcholine during the period examined. About 50% of the total phosphatidylcholine was disaturated which is approximately the same as in adult rabbit lung lavage. The fatty acid composition, however, did change. There was an increase in the amount of 16:0 from about 20% of the total fatty acids in phosphatidylcholine at 27-28 days to about 60% at full term, after birth, and in the adults. There was a corresponding decrease in the amounts of 14:0, 18:0, and longer chain fatty acids, most of which were saturated. In the lavaged lung tissue, there was a 2.6-fold increase in the percentage of phosphatidylcholine which was disaturated during the period 27-31 days' gestation. It had not decrease to the adult value 24 hr after birth.
MATERIALS AND METHODS
INTRODUCTION
The lung is lined with surface-active material, pulmonary surfactant, which is believed to lower surface tension at the air-liquid interface and prevent alveolar collapse (1). Surfactant can be obtained from the lung by lavaging with saline. It has been shown to be largely phospholipid of which phosphatidylcholine is the most abundant component (1,2). Dipalmitoylglycerophosphocholine is believed to be the major surface-active component of pulmonary surfactant (1,2). Insufficient surfactant at birth can lead to
T i m e d p r e g n a n t rabbits (New Zealand W h i t e ) were p u r c h a s e d from Glochester Rabbitry, Chepachet, RI. The doe was sacrificed with intravenous sodium pentobarbital (80 mg/kg). The fetuses were immediately delivered by cesarean section and sacrificed with intraperitoneal sodium pentobarbital (30 rag) prior to breathing, while still in their amniotic sacs. Newborn animals were similarly sacrificed 1-24 hr after delivery by cesarean section, during which time they were kept in an incubator at constant temperature (31 C) and humidity. The lungs were lavaged in situ, via a tracheal cannula, with five 1-3 ml aliquots of 0.9%
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FETAL LUNG PHOSPHATIDYLCHOLINE
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TABLE I Disaturated Phosphatidylcholine Content of Lung Lavage and Lavaged Lung Tissue from Fetal, Newborn, and Adult Rabbits a Lung lavage Lung tissue Disaturated phosphatidylcholine as % of total phosphatidylcholine Fetuses
Newborns
Gestational age (days) 27 28 29 30 31 Period after birth (hours)
49.3±1.9 55.5±0.9 46.0±5.3 50.2±4.4 52.8±3.0
27.0±3.2 34.0±4.5 34.1±3.8 35.2±3.3 41.6±2.6
54.0 ± 1.4
42.2 47.3 57.6 45.8 45.2 28.6
l
3 6 18 24 Adult b
± 1.8 ± 1.5 ± 0.8 ± 3.9 ± 1.7 + 1.3
aThe data are the means (±SE) from 3-6 experiments in each of which lavage and tissue from all fetuses in 1-2 litters were combined. The newborn and adult data are from 3-5 animals. bThe adult data are from a previous study (14). NaC1. The lavage liquid was c e n t i f u g e d at 1000 x g to r e m o v e cellular material. Lipids were ext r a c t e d f r o m the l y o p h i l i z e d lavage a n d tissue as described previously (6,10). P h o s p h a t i d y l c h o l i n e was isolated b y t h i n layer c h r o m a t o g r a p h y (TLC) on Q u a n t u m LQD plates ( Q u a n t u m Industries, Fairfield, N J) in chloroform-methanol-7 M NH4OH (60:35:4, b y vol). It was eluted f r o m t h e gel a n d quantirated b y p h o s p h o r u s assay as described previously (10). P h o s p h a t i d y l c h o l i n e was saponified w i t h m e t h a n o l i c KOH (11). T h e f a t t y acids were m e t h y l a t e d , a n d t h e m e t h y l esters were analyzed b y gas liquid c h r o m a t o g r a p h y as described previously (12). D i s a t u r a t e d p h o s p h a t i d y l c h o l i n e was separated from total phosphatidylcholine from lung tissue f o l l o w i n g o s m i u m t e t r o x i d e t r e a t m e n t b y the m e t h o d of Mason et al. (13) as d e s c r i b e d previously (14). Lavage d i s a t u r a t e d p h o s p h a t i d y l c h o l i n e was similarly m e a s u r e d e x c e p t t h a t it was s e p a r a t e d f r o m t h e oxidized p h o s p h a t i d y l c h o l i n e b y TLC o n Q u a n t u m LQD plates in chloroform-methanol-7 M NH4OH ( 7 0 : 3 0 : 3 . 5 , b y vol) (13). Q u a n t i t a t i o n was b y the u l t r a m i c r o p h o s p h o r u s assay d e s c r i b e d b y B a r t l e t t (15). In this p r o c e d u r e , 86% o f standard dipalmitoylglycerophosphocholine and 1.5% of s t a n d a r d d i o l e o y l g l y c e r o p h o s p h o c h o line were r e c o v e r e d in t h e p h o s p h a t i d y l c h o l i n e band. Standard p h o s p h o l i p i d s were p u r c h a s e d
f r o m Serdary, L o n d o n , O n t a r i o , C a n a d a a n d s t a n d a r d f a t t y acid m e t h y l esters f r o m Supelco, Bellefonte, PA. O t h e r chemicals were r e a g e n t grade or better. R ESU LTS
In t h e lung lavage f r o m fetal rabbits, a b o u t half of t h e t o t a l p h o s p h a t i d y l c h o l i n e was disaturated throughout the developmental period e x a m i n e d ( T a b l e I). A similar a m o u n t of phosp h a t i d y l c h o l i n e is d i s a t u r a t e d in lung lavage f r o m t h e adult rabbit. T h e f a t t y acid c o m p o s i t i o n o f p h o s p h a t i d y l c h o l i n e from fetal a n d n e w b o r n r a b b i t lung lavage is s h o w n in T a b l e II. In a g r e e m e n t w i t h t h e finding w i t h respect to d i s a t u r a t e d p h o s p h a t i d y l c h o l i n e , t h e r e was little change in t h e t o t a l a m o u n t o f s a t u r a t e d f a t t y acids d u r i n g developm e n t . T h e c o m p o s i t i o n of t h e f a t t y acids, h o w ever, did change. T h e r e was an increase in t h e a m o u n t of 16:0 a n d a c o r r e s p o n d i n g decrease in t h e a m o u n t s o f 14:0, 18:0, a n d longer c h a i n ( > 18 c a r b o n a t o m s ) f a t t y acids f r o m 27 days' g e s t a t i o n to full term. T h e f a t t y acid c o m p o s i t i o n of lung lavage p h o s p h a t i d y l c h o l i n e at 31 days' g e s t a t i o n a n d 6 hr a f t e r b i r t h was similar to t h a t of adults (12). A t 31 days' g e s t a t i o n a n d a f t e r b i r t h , as well as in adults (12), 16:0 acc o u n t e d for a b o u t 60% of t h e t o t a l a n d 90% of t h e s a t u r a t e d f a t t y acids. Thus, t h e b u l k o f d i s a t u r a t e d p h o s p h a t i d y l c h o l i n e in l u n g lavage at this d e v e l o p m e n t a l stage m u s t b e dipalmiLIPIDS, VOL. 12, NO. 12
1052
S.A. R O O N E Y A N D L.I. G O B R A N
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L I P I D S , V O L . 12, N O . 12
FETAL LUNG PHOSPHATIDYLCHOLINE
1053
TABLE III Fatty Acid Composition of Phosphatidylcho]ine from Lavaged Lung Tissue of Fetal Rabbits a Fatty acid methyl ester n 14:0 14:2 15:0 16:0 16:1 18:0 18:1 18:2 18:3 22:0 20:4 Others
24 b
Gestational age (days) 28 29
27
30
31
4
3
3
3
3
3
3.3• 1.7• trace 35.9• 7.2• 6.8• 24.9• 1.2 13.9• trace trace 3.4• 1.0 2.9• 1.4
0.8• trace trace 39.2• 4.4• 7.5• 23.8• 14.6• 1.2 1.2• 0.7• 6.1• 1.7•
1.6e0.1 1.7• trace 47.0• 5.2• 5.8 • 17.5• 11.5• 0.9• 0.8• 6.8• 1.2•
2.5• 0.9• 0.6• 49.0• 6.5• 5.6• 15.6• 11.3• 1.1 0.5 • 0.6• 5.7• 1.0•
2.3• 0.6• 0.5• 49.1• 7.2• 4.7• 15.0• 11.6• 0.9• 0.7• 6.5• 0.8•
3.2• trace 0.8• 51.5• 8.4• 4.7• 13.1 • 12.2• 0.8• 0.6• 3.6• 1.4 1.1 •
aTrace, < 0.5%. Other details as in Table II. bThe lungs from the 24 day fetal rabbits were not lavaged prior to lipid extraction. t o y l g l y c e r o p h o s p h o c h o l i n e . (The fatty acid c o m p o s i t i o n of the disaturated species was n o t determined because of the small a m o u n t s of material available for analysis). At 29 and 30 days' gestation, 16:0 a c c o u n t e d for about 50% of the total and 76-77% of the saturated fatty acids so that, even at this stage of d e v e l o p m e n t , d i p a l m i t o y l g l y c e r o p h o s p h o c h o l i n e w o u l d be the m a j o r disturated species. At 27 and 28 days, however, 16:0 a c c o u n t e d for only a b o u t 20% of the total and 30% of the saturated f a t t y acids. Thus, at this d e v e l o p m e n t a l stage, dipalm i t o y l g l y c e r o p h o s p h o c h o l i n e is p r o b a b l y a m i n o r c o m p o n e n t of lung lavage phosphatidylcholine. Long chain fatty acids, largely saturated, a c c o u n t e d for 31% of the total at 27 days' gestation and 12% at 28 days b u t only 2-3% at 29-31 days. Arachidonic acid (20:4), w h i c h is c h a r a c t e r i s t i c a l l y absent from surfactant, accounted for 5% of the total at 27 days. 0.5-1% at 28-30 days, and was not seen at 31 days or after birth. There was an increase in the a m o u n t of 16:1 from 4% of the t o t a l at 27 days to 12% at 28-29 days and to a b o u t 16% at full term. In the lavaged lung tissue, disaturated phosphatidylcholine increase from 27% of the t o t a l phosphatidylcholine at 27 days' gestation to 42% at 31 days (Table I). It increased even further after birth and did not decrease to the adult value during the period examined. The fatty acid c o m p o s i t i o n of p h o s p h a t i d y l c h o l i n e from lavaged lung tissue is shown in Table III. There was little change during the period 24-31 days' gestation other than an increase in the a m o u n t of 16:0 from 36% of the total fatty acids to 52% and a corresponding decrease in the a m o u n t of 18:1 from 25% to 13%. These changes in fatty acid c o m p o s i t i o n are consistent
with the d e v e l o p m e n t a l increase in disaturated phosphatidylcholine. By 31 days' gestation, the fatty acid c o m p o s i t i o n of lung tissue phosphatidylcholine was similar to that of adults (14). DISCUSSION
Surfactant appears to be synthesized by the fetal lung towards the end of gestation (2) and largely stored, presumably in lamellar inclusion bodies in type II alveolar epithelial cells (16), until after birth when it is released in large q u a n t i t y into the alveoli (6,17). It has been shown that there is an increase in the a m o u n t of phospholipid, particularly phosphatidylcholine, in lung lavage during the latter part of gestation in the fetal rabbit (6) and lamb (18). However, the total a m o u n t in the lavage even at full term accounts for no m o r e than 1% of the total p u l m o n a r y phospholipid or phosphatidylcholine (8). One day after birth, on the other hand, 7% of total p u l m o n a r y phospholipid and 10% of total p u l m o n a r y p h o s p h a t i d y l c h o l i n e is recovered in the lavage (8). There are also changes in the c o m p o s i t i o n of lung lavage phospholipid during development. At 27 days' gestation in the rabbit, for instance, phosphatidylcholine accounts for only 29% of the total phospholipid while sphingomyelin accounts for 38% (6). After birth (6) and in the adult (10), phosphatidylcholine accounts for at least 80% of the total but sphingomyelin accounts for less than 3%. Similarly, as shown in this paper, there are changes in the fatty acid c o m p o s i t i o n of p h o s p h a t i d y l c h o l i n e from lung lavage during fetal development. F r o m these studies, it is apparent that dipalmitoylglycerophosphocholine, the major surface-active comp o n e n t of p u l m o n a r y surfactant in adults (1,2), LIPIDS, VOL. 12, NO. 12
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S.A. ROONEY AND L.I. GOBRAN
is only a relatively m i n o r c o m p o n e n t o f lung lavage p h o s p h o l i p i d b e f o r e a b o u t 29 d a y s ' gest a t i o n in the rabbit. It is o f i n t e r e s t t h a t the surface activity o f lung lavage p h o s p h a t i d y l c h o line also begins t o increase at t h e same gestational age (19). There are few previous studies on the f a t t y acid c o m p o s i t i o n o f lung lavage p h o s p h a t i d y l choline during fetal d e v e l o p m e n t . Fijiwara et al. (18) r e p o r t e d t h a t t h e a m o u n t o f 16:0 in p h o s p h a t i d y l c h o l i n e f r o m fetal lamb lung lavage increased w i t h increasing gestational age. F r o m f a t t y acid analysis, these w o r k e r s calculated t h a t the p e r c e n t a g e o f m i n i m u m d i s a t u r a t e d p h o s p h a t i d y l c h o l i n e increased during t h e same period (18). Gluck et al. (20) also r e p o r t e d t h a t t h e r e was m o r e 16:0 in p h o s p h a t i d y l c h o l i n e f r o m lung lavage o f fetal rabbits at 31 days' g e s t a t i o n t h a n in t h a t f r o m t h o s e at 23 days. We have carried o u t studies o n tracheal and gastric liquids f r o m n e w b o r n h u m a n s at b i r t h (sources o f alveolar liquids) and f o u n d t h a t p h o s p h a t i d y l c h o l i n e in the materials from p r e m a t u r e i n f a n t s c o n t a i n e d less 16:0 and m o r e 18 c a r b o n f a t t y acids t h a n in t h a t f r o m full t e r m i n f a n t s (21). Similar data has b e e n rep o r t e d for a m n i o t i c fluid ( a n o t h e r source o f fetal s u r f a c t a n t ) f r o m h u m a n s (22-25) and sheep (26). A n increase in t h e a m o u n t o f p u l m o n a r y d i s a t u r a t e d p h o s p h a t i d y l c h o l i n e during fetal d e v e l o p m e n t has also b e e n r e p o r t e d b y others. Fujiwara et al. (18) and B r u m l e y et al. (27) reported a d e v e l o p m e n t a l increase in the a m o u n t of d i s a t u r a t e d p h o s p h a t i d y l c h o l i n e in lavaged and i n t a c t fetal lamb lung, respectively. S o o d s m a et al. (9) r e c e n t l y r e p o r t e d increases in the a m o u n t s o f d i s a t u r a t e d p h o s p h a t i d y l c h o line and d i p a l m i t o y l g l y c e r o p h o s p h o c h o l i n e in i n t a c t lungs f r o m fetal rabbits during t h e p e r i o d 23.5 to 30.5 d a y s ' gestation. The increased a m o u n t o f d i s a t u r a t e d phosp h a t i d y l c h o l i n e in lavaged lung tissue w i t h increasing g e s t a t i o n a l age m a y reflect increased synthesis and storage o f s u r f a c t a n t prior to birth. It is o f i n t e r e s t t h a t the a m o u n t o f disaturated p h o s p h a t i d y l c h o l i n e in t h e lavaged tissue does n o t decrease i m m e d i a t e l y after b i r t h w h e n t h e r e is a t r e m e n d o u s increase in t h e a m o u n t o f p h o s p h a t i d y l c h o l i n e - a b o u t h a l f o f w h i c h is d i s a t u r a t e d - in t h e lavage (6,17). It is possible t h a t n o t all o f t h e s t o r e d s u r f a c t a n t is released i n t o the alveoli in the first 24 hr after birth. The l o w e r level o f d i s a t u r a t e d p h o s p h a t i d y l c h o line f o u n d in t h e adult lung tissue m a y n o t be r e a c h e d for s o m e t i m e a f t e r birth. ACKNOWLEDGMENT This work was supported by a grant (HD-10192) LIPIDS, VOL. 12, NO. 12
from the National Institute of Child Health and Human Development, USPHS. REFERENCES 1. Goerke, J. Biochim. Biophys. Acta 344:241 (1974). 2. Van Golde, L.M.G., Am. Rev. Respir. Dis. 114:977 (1976). 3. Farrell, P.M., and M.E. Avery, Am. Rev. Respir. Dis. 111:657 (1975). 4. Farrell, P.M., and R.E. Wood, Pediatrics 58:167 (1976). 5. Wu, B., Y. Kikkawa, M.M. Orzalesi, E.K. Motoyama, M. Kaibara, C.J. Zigas, and C.D. Cook, Biol. Neonat 22:161 (1973). 6. Rooney, S.A., T.S. Wai-Lee, L. Gobran, and E.K. Motoyama, Biochim. Biophys. Acta 431:447 (1976). 7. Rooney, S.A., L. Gobran, I. Gross, T.S. Wai-Lee, L.L. Nardone, and E.K. Motoyama, Biochim. Biophys. Acta 450:121 (1976). 8. Rooney, S.A., and E.K. Motoyama, in "Pulmonary Macrophage and Epithelial Cells," 16th Annual Hanford Biology Symposium, Energy Research and Development Administration Conference 760927, 1977, p. 162. 9. Soodsma, J.F., L.C. Mims, and R.D. Harlow, Biochim. Biophys. Acta 424:159 (1976). 10. Rooney, S,A., P.M. Canavan, and E.K. Motoyama, Biochim. Biophys. Acta 360:56 (1974). 11. Rooney, S.A., and H. Goldfine, J. Bacteriol. 111:531 (1972). 12. Rooney, S.A., B.A. Page-Roberts, and E.K. Motoyama, J. Lipid Res. 16:418 (1975). 13. Mason, R.J., J. Nellengogen, and J.A. Clements, J. Lipid Res. 17:281 (1976). 14. Rooney, S.A., L.L. Nardone, D.L. Shipiro, E.K. Motoyama, L. Gohran, and N. Zaehringer, Lipids 12:438 (1977). 15. Bartlett. G.R., J. Biol. Chem. 234:466 (1959). 16. Rooney, S.A., in "Lung Cells in Disease," Edited by A. Bouhuys, North Holland, Amsterdam, The Netherlands, 1976, p. 147. 17. Rooney, S.A., L.I. Gobran, and T.S. Wai-Lee, J. Clin. Invest. 60:754 (1977). 18. Fujiwara, T., F.H. Adams, S. Sipos, and A. EI-Salawy, Am. J. Physiol. 215:375 (1968). 19. Gluck, L., E.K. Motoyama, H.L. Smits, and M.V. Kulovich, Pediatr. Res. 1:237 (1967). 20. Gluck, L., R.A. Landowne, and M.V. Kulovich, Pediatr. Res. 4:352 (1970). 21. Motoyama, E.K., Y, Namba, and S.A. Rooney, Clin. Chim. Acta 70:449 (1976), 22. Ekelund, L., A. Gosta, and B. Astedt, J. Obstet. Gynaecol. Br. Commonw. 80:912 (1973). 23. Roux, J.F., J. Nakamura, and M. Frosolono, Am. J. Obstet. Glynecol. 119:838 (1974). 24. Frantz, T., T. Lindback, J. Skjaeraasen, and S. Graven, Acta Obstet. Gynecol. Scand. 54:33 (1975). 25, Schirar, A., J.P. Vielh, L.G. Alcindor, and J.P. Gautray, Am. J. Obstet. Gynecol. 121:653 (1975). 26, Ogawa, Y., Tohoku J. Exp. Med. 108:307 (1972). 27. Brumley, G.W., V. Chernick, W.A. Hodson, C. Normand, A. Fenner, and M.E. Avery, J. Clin. Invest. 46:863 (1967).
[Received July 18, 1977]
