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Fetal lamb lung phosphatidylcholine: Response to asphyxia and recovery
October 1980
The Journal o f P E D I A T R I C S
631
Fetal lamb lung phosphatidylcholine: Response to asphyxia and recovery Acute fetal asphyxia resulting from maternal blood loss and hypotension causes a reduction in the incorporation of precursors into disaturated phosphatidyleholine, the principal lipid in the pulmonary surfaetant. Treatment of the maternal hypotension is associated with return of fetal lung DSPC synthesis to control levels by 72 hours.
George W. Brumley, M.D.,* and Carlyle Crenshaw, M.D., D u r h a m , N. JC.
INTRAUTERINE PULMONARY INJURY has been considered to be a primary cause of pulmonary surfactant deficiency, which results in the respiratory distress syndrome of infancy.1-3 It has been assumed that fetal asphyxia and the associated reduction in pulmonary blood flow4-~ result in pulmonary ischemia and in compromise of the synthesis of disaturated phosphatidylchofine, the principal lipid in pulmonary surfactant. This study was performed to determine if hypotensive insults, similar to those experienced under clinical conditions, result in changes in fetal lung DSPC and whether recovery from such insults could occur in utero.
MATERIALS AND M E T H O D S Fifteen (15) dated pregnant ewes (with single fetuses) at 122 days' gestation, under spinal anesthesia with local anesthesia for the fetus, were prepared with maternal and fetal umbilical arterial catheters and then were allowed to recover from the effects of surgery. At 125 days' gestation the maternal and fetal blood pressures, heart rates, and acid base status were monitored, and seven (7) unanesthetized and free-standing ewes were bled through a large jugular vein catheter into heparinized bags. Sufficient maternal blood was removed to produce mean maternal arterial blood pressures approximately one-half prephlebotomy levels, following which the fetuses became acidotFrom the Division of Perinatal Medicine, Departments of Pediatrics, Obstetrics, and Gynecology, Duke University Medical Center. Supported in part by a grant from the National Heart, Lung and Blood Institute (HD-06311). *Reprint address: Box 3967, Division of Perinatal Medicine. Duke University Medical Center, Durham, NC 27710.
0022-3476/80/100631 +04500.40/0 9 1980 The C. V. Mosby Co.
ic. Thereafter, the fetuses developed tachycardia and hypertension, followed by bradycardia and hypotension. The maternal hypotension was maintained for 30 minutes after the fetus became acidotic. Four fetuses ("acute stress") were delivered and killed for immediate biochemical study. Three ewes were transfused with the blood previously removed, which allowed the ewe and her in utero fetus to recover ("stress-recovered"). These latter animals were killed 72 hours later, at 128 days' gestation. Eight nonphlebotomized lambs (five at 125 days and three at 128 days) were delivered, killed, and their lungs used as controls. Abbreviations used DSPC: disaturated phosphatidylcholine PC: phosphatidylcholine RDS: respiratory distress syndrome DNA: deoxyribose nucleic acid The lungs were removed and samples weighing approximately 0.5 gm were sliced thin and incubated with 6 ml modified Ringer's solution pH 7.4 containing 30 ~Ci carrier free (32p) orthophosphate (New England Nuclear, Boston) and 10/LCi 1-(14C)-patmitic acid, 49.2 mCi/mmol (New England Nuclear, Boston) which was complexed to human serum albumin. The lung slices were incubated in a shaking water bath at 37~ with a gas phase of 100% hydrated oxygen, as previously described. ~ The reaction was terminated by the addition of 10 ml methanol and the tissue slices were homogenized in a Potter tube with a Teflon pestle. Chloroform (20 ml) was added and the resultant chloroform:methanol solvent used to extract the lipids, as described by Folch et al. 7 The lipid extract was washed and the resultant extract analyzed for content of
VoL 97, No. 4, pp. 631-634
632
Brumley and Crenshaw
The Journal of Pediatrics October 1980
Table I. Physiologic measurements from control, stressed, and stressed-recovered fetal sheep arterial b l o o d
Type
Gestational age
pH
.
Pco~
HCO~
Po~
Hematocrit
Stress (4)
125
6.83 • 0.08
60 -+ 5
9 + 2
18 _+2
33
Control (4) Stress (3)
125
7.32 -+ 0.05 6.96 _+ 0.06
47 -+ 4 42 + 7
23 _+ 1 10 _+ 2
22 _+5 19 _+6
39 _+3 39 _+1
Recovered (3) Control
128
7.33 _+ 0.01 7.36
53 _+ ! 49
26 _+ 1 25
18 _+3 20
35 _+3
Acute stress study
125
Stress:recovery study
128
(2) * = P < 0.005. ( ) = Number of animals.
Table II. Lung phosphatidylchofine from control and asphyxiated fetal lambs (acute-125 days gestation)
Control
Stressed
t~g PC
fig D S P C
[32p]
mg DNA
mg DNA
fig PC
695 _+ 15 (5) 806 _+ 61 (4) NS
t35 +- 16 (4) 187 _+ 32 (3) NS
1,386 _+ 162
(5) 1,165 __ 230
(4) NS
[,4C]
[,,c]
fig DSPC
I~g PC
I~g DSPC
932 + 86 (3) 512 _+ 34 (3) P < 0.01
861 _+ 35
(5) 672 _+ 57
(4) P < 0.02
1,438 _+ 82 (4) 901 _+ 69 (3) P < 0.005
] = D i s i n t e g r a t i o n p e r m i n u t e p e r isotope. ) = N u m b e r a n i m a l s s t u d i e d in.duplicate.
phosphatidylcholine and disaturated phosphatidylcholine. using the thin layer chromatographic method described by Skipski et al ~ and the cryochromatographic method described by Henderson and Clayton. 9 The resultant phosphotipids were quantitated by analyzing the inorganic phosphorus content as described by Bartlett, TM and the radioactivitv estimated using scintillation spectrometry and quench correction factors derived by the external standard method. Deoxyribose nucleic acid was determined on the tissue residue following lipid extraction by the method o f Schneider. I~ Statistical analysis of the data was performed using the "t statistic for two means" program described b y Brownlee? ~
RESULTS Table I indicates that the acute-stressed lambs received a significant asphyxial insult, presumably from placental hypoperfusion. In general, the lowest Pa% values (range 10 to31 X 14:4 _+ 6.8 SEM vs range 11 to 40 X 21.6 • 3.8
SEM m m Hg) were reached in the stressed animals prior to the lowest pH, although these values were not significantly lower than in controls. The Pa% values in Table I were determined at the same time as the pH. At the time of sacrifice, the acid base status of the stress-recovered ewes and fetuses had returned to normal. Evidence of the prior stress remained, however, in that the amnionic fluid was gelatinous and laden with meconium. The control and acute-stressed lambs at 125 days' gestation showed no significant difference in the concentration of phosphatidylcholine or disaturated phosphatidylcholine (Table II). Although there was no detectable reduction in the incorporation of (~2p) into PC, when D S P C was analyzed, the (3~p) specific activities were significantly lower. Both PC and D S P C (x4C) palmitic acid specific activities were reduced significantly as compared to those of controls. Lung phospholipids from stress-recovered lambs which were allowed to remain in utero for three days after their
Volume 97 Number 4
asphyxial insult and from 128 day controls are shown in Table III. The content of DSPC wa s not different in the stress-recovered and control lungs. No differences were evident in the incorporation of (32p) into PC or of (14C) palmitate into DSPC in the stress-recovered animals. (32p) incorporation into DSPC was not determined in these animals because of technical difficulties.
Fetal lamb phosphatidylcholine
Table III. Lung phosphatidylcholine from control and asphyxiated fetal lambs after 3 days recovery in utero (128 days' gestation) fig PC
[a2p]
fig DSPC
['~C]
mg DNA
Control
DISCUSSION The fetal lamb lung at 125 days' gestation has just begun the anatomical and biochemical transition which is necessary for air-breathing and extrauterine life. This gestational age was chosen for study because it is somewhat similar to that found in the preterm infant who most oftenhas RDS. The significant increase (P < 0.005) in the concentration of DSPC in the fetal lamb lung observed between 125 days and 128 days reflects the increases in type :II alveolar epithelial cell lamellar bodies normally observed during lung maturation. 13 Prior studies by Reynolds and coworkers TM have shown that lambs of 130 to 136 days' gestation develop RDS and abnormal surface tension measurements following prenatal asphyxia. Adams et aP 5 demonstrated no change in lung lipid content, composition, or surface activity using a similar, but somewhat more mature fetal lamb model. Sundell and Stahlman 16 have shown that pharmacologically induced hypotension in the ewe causes RDS in the newborn preterm lamb and, more recently, Merritt and FerrelW have shown that acidosis reduces the synthesis of fetal rat lung PC in vitro. Sheldon and coworkers 18 have demonstrated that lavage of nonacidotic fetal lambs with amnionic fluid caused a reduction in de novo synthesis of lung PC when studied in vitro. Preliminary evidence by these latter authors from a limited number of lavaged lambs which were also acidotic indicated that in the presence of acidosis, other non-de novo PC synthetic pathways also were compromised. Maternal hypotension in our acute studies was associated with biochemical evidence of significant reduction in incorporation of precursors into fetal lung DSPC and, therefore, a reduction in DSPC specific activity. This suggested that DSPC specific activity, in the absence of a change in the lung DSPC pool size, was a sensitive index of DSPC synthesis and may correlate well with clinical RDS. Since (a2p) incorporation reflects de novo synthesis, these data indicate that asphyxial insult had an impact on the synthesis of the basic PC molecule prior to the fatty acid rearrangement which produces the DSPC molecule characteristic of the pulmonary surfactant lipid. Although (14C) palmitate incorporation also reflects de novo synthesis, it has been shown in the rat by Moriya and
633
803 • 80
(3) Stressed
812 _+ 65
(3) NS
256 • 47 (3) 259 • 36 (3) NS
[,,c] fig DSPC
•
951 99
179 +_ 52
(3)
(3)
1,221 • 262
184
(3)
(2)
NS
2,277 _+ 526 (3) 2,603 + 368 (3) NS
[ ] = Disintegrationper minute per isotope. ( ) = Number animals studied in duplicate. Knoh 19that de novo synthesis contributes less than 17% of the total DSPC synthesized by the lung. Therefore, it is more likely that the reduction in ('~C) palmitate incorporation reflects compromise in the phospholipase A ~ acyltransferase pathway, which is responsible for nonsurfactant PC fatty acid rearrangement to the disaturated species. Thus, there appears to be suggestive evidence that both the de novo and the rearrangement pathways are compromised temporarily by asphyxia. The stress-recovered fetal lambs demonstrated a number of additional points: (1) profound acidosis of short duration was not lethal, (2) reinstatement of maternal blood pressure apparently permitted return of adequate placental blood flow and repair 9f the fetal acidosis, (3) the amnionic fluid in these Iambs was contaminated heavily with meconium and had become gelatinous in character, and (4) cross-section of the lung demonstrated meconium-containing fluid in the most peripheral portion of the lung, indicating that the fetal asphyxia had induced significant and sustained ventilation of the meconiumcontaining amnionic fluid. The comparison of the biochemical measurements in the control and in stressrecovered lamb lungs also contributed to our understanding of the response of the fetus to such insults: (1) no apparent, sustained compromise in DSPC synthesis occurred, since the concentration of DSPC in the 128-day lamb was not decreased in the stress-recovered lamb as compared to that in 128-day controls, and the concentration of DSPC wa s significantly higher than in the 125-day lambs, suggesting that normal accumulation of DSPC had continued after recovery from the asphyxial insult; and (2) no evidence of compromise in synthetic activity remained in that the incorporation of radioactive precursors was comparable in the stress-recovered and control animals. Although the intrauterine asphyxial model simulates the complex events which compromise the lung when the
634
Brumley and Crenshaw
fetus is asphyxiated, and demonstrates t h a t the synthesis of a critical c o m p o n e n t o f the pulmonary surfactant is compromised, it does not discern whether amnionic fluid (with or without meconium) or ischemia, is the offending agen t. Thus, asphyx!al injury t o the lung surfactant system appears to be a complex insult which involves a direct effect of ammi0nic fluid and additional compromise by acidosis. Additional studies o f surfactant D S P C under asphyxial conditions with protection of the lung from aspiration will be necessary to separate clearly the effects of ammionic fluid and acidosis on the lung, and to demonstrate Milch factor may be etiologicaily important in RDS. The authors appreciate the significant technical assistance of Ms. Betty Tuggle and Mr. Lawrence Kodack, and the assistance of Ms, Camilla Vestal in the preparation of the manuscript. REFERENCES
1. Avery ME, and Mead J: Surface properties in relation to atelectasis and hyaline membrane disease, Am J Dis Child 97:517, 1959. 2. Chu J, Clements J, Cotton E, Klaus M, Sweet A, Thomas A, and Tooley W: The pulmonary hypoperfusion syndrome, Pediatrics 35:373, 1965. 3. Brumtey G, Hodson A, and Avery M: Lung phospholipids and surface tension correlations in infants with and without hyaline membrane disease and in adults, Pediatrics 40:13, 1967. 4. Berhman R, Lee M, Peterson E, et al: Distribution of the circulation in the normal and asphyxiated primate, Am J Obstet Gynecol 108:956, 1970. 5. Peters L, Sheldon R, Jbnes M, Makowski E, and Meschia G: Blood flow to fetal organs as a function of arterial oxygen content, Am J Obstet Gynecol 135:637, 1979. 6. Brumley G, Knelson J, Schomberg D; and Crenshaw C: Wholeand disaturated lung phosphatidylcholine in cortisol-treated, intrauterine growth retarded and twin control lambs at different gestational ages, Biol Neonate 31:155, 1977.
The Journal of Pediatrics October 1980
7. Folch J, Ascoli J, Lees M, Heath J, and LeBaron F: Preparation of fipid extract from brain tissue, J Biol Chem 191:833, 1951. 8. Skipski V, Peterson L, Sanders J, and Barclay M: Thin layer chromatography of phospholipids using silicic gel without calcium sulphate binder, J Lipid Res 4:227, 1963. 9. Henderson R, and Clayton M: Cryochromatography: a method for the separation of lung phosphoglycerides according to the number and length of saturated fatty acid components, Anal Biochem 70:440, t976. 10. Bartlett G: Phosphorus assay in column chromatography, J Biol Chem 234:466, 1959. 11. Schneider W: Determination of nucleic acids in tissues by pentose analysis in Colowick SP, and Kaplan NP, editors: Methods in enzymology, vol 3, New York, 1957, Academic Press, Inc. p 680. 12. Brownlee K: Statistical theory and methodology in science and eng!neering, New York, 1965, John Wiley and Sons, Inc. 13. Brumley G, Chernick V, Hodson A, et al: Correlations of mechanical stability, morphology, pulmonary surfactant, and phospholipid content in the developing lamb lung, J Clin Invest 46:863, 1967. 14. Reynolds E, Jacobson H, Motoyama E, Kikkawa M, Craig J, Orzalesi M, and Cook C: The effect of immaturity and prenatal asphyxia on the lungs and pulmonary function of newborn Iambs: the experimental production of respiratory distress, Pediatrics 35:382, 1965. 15. Adams F, Nozaki M, Chida N, and Salawy A: Effects of hypoxemia, hypercarbia, acidosis and reduced pulmonary blood flow on the surfactant of fetal lamb lung, J PEDIATR 71:396, 1967. 16. Sundell H, and Stahlman M: Personal communication. 17. Merritt T, and Ferrell P: Diminished pulmonary lecithin synthesis in acidosis: experimental findings as related to the respiratory distress syndrome, Pediatrics 57:32, 1976. 18. Sheldon G, Brazy J, Tuggle B, Crenshaw C, and Brumley G: Fetal lamb lung lavage and its effect on lung phosphatidylcholine , Pediatr Res 13:599, 1979. 19. Mori)a T, and Knoh H: In vivo studies on the de novo synthesis of molecular species of rat lung lecithin, Tohoku J Exp Med 112:241, 1974.
The Journal o f P E D I A T R I C S
631
Fetal lamb lung phosphatidylcholine: Response to asphyxia and recovery Acute fetal asphyxia resulting from maternal blood loss and hypotension causes a reduction in the incorporation of precursors into disaturated phosphatidyleholine, the principal lipid in the pulmonary surfaetant. Treatment of the maternal hypotension is associated with return of fetal lung DSPC synthesis to control levels by 72 hours.
George W. Brumley, M.D.,* and Carlyle Crenshaw, M.D., D u r h a m , N. JC.
INTRAUTERINE PULMONARY INJURY has been considered to be a primary cause of pulmonary surfactant deficiency, which results in the respiratory distress syndrome of infancy.1-3 It has been assumed that fetal asphyxia and the associated reduction in pulmonary blood flow4-~ result in pulmonary ischemia and in compromise of the synthesis of disaturated phosphatidylchofine, the principal lipid in pulmonary surfactant. This study was performed to determine if hypotensive insults, similar to those experienced under clinical conditions, result in changes in fetal lung DSPC and whether recovery from such insults could occur in utero.
MATERIALS AND M E T H O D S Fifteen (15) dated pregnant ewes (with single fetuses) at 122 days' gestation, under spinal anesthesia with local anesthesia for the fetus, were prepared with maternal and fetal umbilical arterial catheters and then were allowed to recover from the effects of surgery. At 125 days' gestation the maternal and fetal blood pressures, heart rates, and acid base status were monitored, and seven (7) unanesthetized and free-standing ewes were bled through a large jugular vein catheter into heparinized bags. Sufficient maternal blood was removed to produce mean maternal arterial blood pressures approximately one-half prephlebotomy levels, following which the fetuses became acidotFrom the Division of Perinatal Medicine, Departments of Pediatrics, Obstetrics, and Gynecology, Duke University Medical Center. Supported in part by a grant from the National Heart, Lung and Blood Institute (HD-06311). *Reprint address: Box 3967, Division of Perinatal Medicine. Duke University Medical Center, Durham, NC 27710.
0022-3476/80/100631 +04500.40/0 9 1980 The C. V. Mosby Co.
ic. Thereafter, the fetuses developed tachycardia and hypertension, followed by bradycardia and hypotension. The maternal hypotension was maintained for 30 minutes after the fetus became acidotic. Four fetuses ("acute stress") were delivered and killed for immediate biochemical study. Three ewes were transfused with the blood previously removed, which allowed the ewe and her in utero fetus to recover ("stress-recovered"). These latter animals were killed 72 hours later, at 128 days' gestation. Eight nonphlebotomized lambs (five at 125 days and three at 128 days) were delivered, killed, and their lungs used as controls. Abbreviations used DSPC: disaturated phosphatidylcholine PC: phosphatidylcholine RDS: respiratory distress syndrome DNA: deoxyribose nucleic acid The lungs were removed and samples weighing approximately 0.5 gm were sliced thin and incubated with 6 ml modified Ringer's solution pH 7.4 containing 30 ~Ci carrier free (32p) orthophosphate (New England Nuclear, Boston) and 10/LCi 1-(14C)-patmitic acid, 49.2 mCi/mmol (New England Nuclear, Boston) which was complexed to human serum albumin. The lung slices were incubated in a shaking water bath at 37~ with a gas phase of 100% hydrated oxygen, as previously described. ~ The reaction was terminated by the addition of 10 ml methanol and the tissue slices were homogenized in a Potter tube with a Teflon pestle. Chloroform (20 ml) was added and the resultant chloroform:methanol solvent used to extract the lipids, as described by Folch et al. 7 The lipid extract was washed and the resultant extract analyzed for content of
VoL 97, No. 4, pp. 631-634
632
Brumley and Crenshaw
The Journal of Pediatrics October 1980
Table I. Physiologic measurements from control, stressed, and stressed-recovered fetal sheep arterial b l o o d
Type
Gestational age
pH
.
Pco~
HCO~
Po~
Hematocrit
Stress (4)
125
6.83 • 0.08
60 -+ 5
9 + 2
18 _+2
33
Control (4) Stress (3)
125
7.32 -+ 0.05 6.96 _+ 0.06
47 -+ 4 42 + 7
23 _+ 1 10 _+ 2
22 _+5 19 _+6
39 _+3 39 _+1
Recovered (3) Control
128
7.33 _+ 0.01 7.36
53 _+ ! 49
26 _+ 1 25
18 _+3 20
35 _+3
Acute stress study
125
Stress:recovery study
128
(2) * = P < 0.005. ( ) = Number of animals.
Table II. Lung phosphatidylchofine from control and asphyxiated fetal lambs (acute-125 days gestation)
Control
Stressed
t~g PC
fig D S P C
[32p]
mg DNA
mg DNA
fig PC
695 _+ 15 (5) 806 _+ 61 (4) NS
t35 +- 16 (4) 187 _+ 32 (3) NS
1,386 _+ 162
(5) 1,165 __ 230
(4) NS
[,4C]
[,,c]
fig DSPC
I~g PC
I~g DSPC
932 + 86 (3) 512 _+ 34 (3) P < 0.01
861 _+ 35
(5) 672 _+ 57
(4) P < 0.02
1,438 _+ 82 (4) 901 _+ 69 (3) P < 0.005
] = D i s i n t e g r a t i o n p e r m i n u t e p e r isotope. ) = N u m b e r a n i m a l s s t u d i e d in.duplicate.
phosphatidylcholine and disaturated phosphatidylcholine. using the thin layer chromatographic method described by Skipski et al ~ and the cryochromatographic method described by Henderson and Clayton. 9 The resultant phosphotipids were quantitated by analyzing the inorganic phosphorus content as described by Bartlett, TM and the radioactivitv estimated using scintillation spectrometry and quench correction factors derived by the external standard method. Deoxyribose nucleic acid was determined on the tissue residue following lipid extraction by the method o f Schneider. I~ Statistical analysis of the data was performed using the "t statistic for two means" program described b y Brownlee? ~
RESULTS Table I indicates that the acute-stressed lambs received a significant asphyxial insult, presumably from placental hypoperfusion. In general, the lowest Pa% values (range 10 to31 X 14:4 _+ 6.8 SEM vs range 11 to 40 X 21.6 • 3.8
SEM m m Hg) were reached in the stressed animals prior to the lowest pH, although these values were not significantly lower than in controls. The Pa% values in Table I were determined at the same time as the pH. At the time of sacrifice, the acid base status of the stress-recovered ewes and fetuses had returned to normal. Evidence of the prior stress remained, however, in that the amnionic fluid was gelatinous and laden with meconium. The control and acute-stressed lambs at 125 days' gestation showed no significant difference in the concentration of phosphatidylcholine or disaturated phosphatidylcholine (Table II). Although there was no detectable reduction in the incorporation of (~2p) into PC, when D S P C was analyzed, the (3~p) specific activities were significantly lower. Both PC and D S P C (x4C) palmitic acid specific activities were reduced significantly as compared to those of controls. Lung phospholipids from stress-recovered lambs which were allowed to remain in utero for three days after their
Volume 97 Number 4
asphyxial insult and from 128 day controls are shown in Table III. The content of DSPC wa s not different in the stress-recovered and control lungs. No differences were evident in the incorporation of (32p) into PC or of (14C) palmitate into DSPC in the stress-recovered animals. (32p) incorporation into DSPC was not determined in these animals because of technical difficulties.
Fetal lamb phosphatidylcholine
Table III. Lung phosphatidylcholine from control and asphyxiated fetal lambs after 3 days recovery in utero (128 days' gestation) fig PC
[a2p]
fig DSPC
['~C]
mg DNA
Control
DISCUSSION The fetal lamb lung at 125 days' gestation has just begun the anatomical and biochemical transition which is necessary for air-breathing and extrauterine life. This gestational age was chosen for study because it is somewhat similar to that found in the preterm infant who most oftenhas RDS. The significant increase (P < 0.005) in the concentration of DSPC in the fetal lamb lung observed between 125 days and 128 days reflects the increases in type :II alveolar epithelial cell lamellar bodies normally observed during lung maturation. 13 Prior studies by Reynolds and coworkers TM have shown that lambs of 130 to 136 days' gestation develop RDS and abnormal surface tension measurements following prenatal asphyxia. Adams et aP 5 demonstrated no change in lung lipid content, composition, or surface activity using a similar, but somewhat more mature fetal lamb model. Sundell and Stahlman 16 have shown that pharmacologically induced hypotension in the ewe causes RDS in the newborn preterm lamb and, more recently, Merritt and FerrelW have shown that acidosis reduces the synthesis of fetal rat lung PC in vitro. Sheldon and coworkers 18 have demonstrated that lavage of nonacidotic fetal lambs with amnionic fluid caused a reduction in de novo synthesis of lung PC when studied in vitro. Preliminary evidence by these latter authors from a limited number of lavaged lambs which were also acidotic indicated that in the presence of acidosis, other non-de novo PC synthetic pathways also were compromised. Maternal hypotension in our acute studies was associated with biochemical evidence of significant reduction in incorporation of precursors into fetal lung DSPC and, therefore, a reduction in DSPC specific activity. This suggested that DSPC specific activity, in the absence of a change in the lung DSPC pool size, was a sensitive index of DSPC synthesis and may correlate well with clinical RDS. Since (a2p) incorporation reflects de novo synthesis, these data indicate that asphyxial insult had an impact on the synthesis of the basic PC molecule prior to the fatty acid rearrangement which produces the DSPC molecule characteristic of the pulmonary surfactant lipid. Although (14C) palmitate incorporation also reflects de novo synthesis, it has been shown in the rat by Moriya and
633
803 • 80
(3) Stressed
812 _+ 65
(3) NS
256 • 47 (3) 259 • 36 (3) NS
[,,c] fig DSPC
•
951 99
179 +_ 52
(3)
(3)
1,221 • 262
184
(3)
(2)
NS
2,277 _+ 526 (3) 2,603 + 368 (3) NS
[ ] = Disintegrationper minute per isotope. ( ) = Number animals studied in duplicate. Knoh 19that de novo synthesis contributes less than 17% of the total DSPC synthesized by the lung. Therefore, it is more likely that the reduction in ('~C) palmitate incorporation reflects compromise in the phospholipase A ~ acyltransferase pathway, which is responsible for nonsurfactant PC fatty acid rearrangement to the disaturated species. Thus, there appears to be suggestive evidence that both the de novo and the rearrangement pathways are compromised temporarily by asphyxia. The stress-recovered fetal lambs demonstrated a number of additional points: (1) profound acidosis of short duration was not lethal, (2) reinstatement of maternal blood pressure apparently permitted return of adequate placental blood flow and repair 9f the fetal acidosis, (3) the amnionic fluid in these Iambs was contaminated heavily with meconium and had become gelatinous in character, and (4) cross-section of the lung demonstrated meconium-containing fluid in the most peripheral portion of the lung, indicating that the fetal asphyxia had induced significant and sustained ventilation of the meconiumcontaining amnionic fluid. The comparison of the biochemical measurements in the control and in stressrecovered lamb lungs also contributed to our understanding of the response of the fetus to such insults: (1) no apparent, sustained compromise in DSPC synthesis occurred, since the concentration of DSPC in the 128-day lamb was not decreased in the stress-recovered lamb as compared to that in 128-day controls, and the concentration of DSPC wa s significantly higher than in the 125-day lambs, suggesting that normal accumulation of DSPC had continued after recovery from the asphyxial insult; and (2) no evidence of compromise in synthetic activity remained in that the incorporation of radioactive precursors was comparable in the stress-recovered and control animals. Although the intrauterine asphyxial model simulates the complex events which compromise the lung when the
634
Brumley and Crenshaw
fetus is asphyxiated, and demonstrates t h a t the synthesis of a critical c o m p o n e n t o f the pulmonary surfactant is compromised, it does not discern whether amnionic fluid (with or without meconium) or ischemia, is the offending agen t. Thus, asphyx!al injury t o the lung surfactant system appears to be a complex insult which involves a direct effect of ammi0nic fluid and additional compromise by acidosis. Additional studies o f surfactant D S P C under asphyxial conditions with protection of the lung from aspiration will be necessary to separate clearly the effects of ammionic fluid and acidosis on the lung, and to demonstrate Milch factor may be etiologicaily important in RDS. The authors appreciate the significant technical assistance of Ms. Betty Tuggle and Mr. Lawrence Kodack, and the assistance of Ms, Camilla Vestal in the preparation of the manuscript. REFERENCES
1. Avery ME, and Mead J: Surface properties in relation to atelectasis and hyaline membrane disease, Am J Dis Child 97:517, 1959. 2. Chu J, Clements J, Cotton E, Klaus M, Sweet A, Thomas A, and Tooley W: The pulmonary hypoperfusion syndrome, Pediatrics 35:373, 1965. 3. Brumtey G, Hodson A, and Avery M: Lung phospholipids and surface tension correlations in infants with and without hyaline membrane disease and in adults, Pediatrics 40:13, 1967. 4. Berhman R, Lee M, Peterson E, et al: Distribution of the circulation in the normal and asphyxiated primate, Am J Obstet Gynecol 108:956, 1970. 5. Peters L, Sheldon R, Jbnes M, Makowski E, and Meschia G: Blood flow to fetal organs as a function of arterial oxygen content, Am J Obstet Gynecol 135:637, 1979. 6. Brumley G, Knelson J, Schomberg D; and Crenshaw C: Wholeand disaturated lung phosphatidylcholine in cortisol-treated, intrauterine growth retarded and twin control lambs at different gestational ages, Biol Neonate 31:155, 1977.
The Journal of Pediatrics October 1980
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