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Alpha1-antitrypsin in amniotic fluid and cord blood of preterm infants with the respiratory distress syndrome
FETAL AND M E D IC IN E
NEONATAL RichardE. Behrman,
Editor
Alphal-antitrypsin in amniotic fluid and cord blood of preterm infants with the respiratory distress syndrome Total protein, alphal-antitrypsin, and alpha2-macroglobulin were measured on amniotic fluid in 125 pregnancies between 11 and 42 weeks" gestation, and on the cord blood o f 66 newborn infants. Amniotic fluid surface active material was assessed by the foam stability test. Amniotic fluid alphal-antitrypsin is linearly and directly related to amniotic fluid total protein (r = O. 703, p < 0.001). The cord alphalantitrypsin is also linearly related to cord total protein, lntrapartum complication s are associated with a significant lowering o f the cord alphal-antitrypsin, lnfants with a negative foam stability test had R D S regardless of the amniotic fluid alpha,-antitrypsin concentration. However, R D S occurred in several infants with an intermediate or positive foam stability test; and this combination was generally associated with an increase o f amniotic fluid alphal-antitrypsin. The cord blood alphal-antitrypsin value did not appear to be related to amniotic fluid surface active material There were 23 infants with cord alphalantitrypsin of less than 0.2 gm/dl and with an intermediate or positive foam stability test; 19 o f them had respiratory difficulties o f varying severity. It is conceivable that infants', in spite o f apparent adequate prenatal lung surfactant, develop respiratory disturbances on the basis of pulmonary fluid and protein transudation and~or reduction or inhibition o f pulmonary surfactant incident to intrapartum complications.
Allan D. Singer, M.D.,* Donald W. Thibeault, M.D.,** Calvin J. Hobel, M.D., and Douglas C. Heiner, M.D., Ph.D., Torrance, Calif.
THE SERUM PROTEINASE INHIB!TORS, alphal-antitrypsin and alpha~,macroglobulin, have been shown to be decreased in the cord blood of infants who subsequently develop RDS. 1-~ Indeed, the outcome of infants with the respiratory distress syndrome may be predicted at birth from the umbilical cord serum levels of the serum trypsin inhibitor capacity. 1 Since the amniotic fluid surface active material is also highly predictive of the risk of a newborn infant to develop RDS, it would be of interest to explore the relationship of amniotic fluid surface material to cord From the Divisions of Perinatal Medicine and Immunology-A llergy, Departments o f Pediatrics and Obstetrics, Harbor General Hospital, UCLA School o f Medicine, *Supported in part by a grant from Hoechst Pharmaceuticals, Inc., Somerville, N. J. **Reprint address: Department of Pediatrics, Harbor General Hospital, 1000 West Carson St., Torrance, Calif. 90509.
and amniotic fluid alphacantitrypsin> 8 To our knowledge, a systematic study of amniotic fluid alphaxantitrypsin throughout gestations and of its relationship to amniotic fluid surface material has not been made> 9. ,o Abbreviations used al-AT: alphal-antitrypsin a2-M: alpha2-macroglobulin RD: respiratorydistress FST: foam stability test TP: total protein cord: umbilical cord It is the purpose of this communication to report studies regarding the concentrations of cord and amniotic fluid alphal-antitrypsinin normal and complicated pregnancies and the relationship of these concentrations to amniotic fluid surface active material. The Journal o f P E D I A T R IC S Vol. 88, No. 1, pp. 87-93
87
88
Singer et al.
The Journal of Pediatrics January 1976
Table I. Intrapartum factors* considered to be detrimental to the fetus in 66 high-risk pregnancies
I No. Five minute apgar score --<6"~ Intrapartum bleeding Breech delivery -<33 weeks' gestation Twins delivered vaginally --<33weeks Maternal hypotension Seizures (eclampsia) Type II deceleration fetal ECG pattern Diabetic ketoacidosis Prolonged second stage labor >2 hours
13 3 3 2 5 1 4 1 1
*Some subjects had more than one factor. t A low apgar score is a postnatal factor strongly suggestive o f an ill fetus.
MATERIAL
AND METHODS
Total protein, albumin, al-AT, a~-M, and surface active material were measured in amniotic fluids of 125 pregnancies of 11 to 42 weeks' gestation. Amniotic fluid was obtained by transabdominal amniocentesis or needle aspiration through the bulging forewaters. Sixty-seven samples of amniotic fluid were obtained within 24 hours of delivery; 15 were prior to therapeutic abortion, and the remaining 43 samples were obtained 24 hours or more prior to delivery. In 44 pregnancies maternal venous blood was obtained at the time of amniotic fluid collection for a~-AT and a~-M measurement. Total protein, albumin, and a1-AT in the cord serum were measured in 66 infants, but a2-M was measured only in 53. The gestational ages of the infants ranged from 17 to 40 weeks. In 35 of these pregnancies, amniotic fluid was also obtained within 24 hours of delivery and studied as above. Sixty-six pregnancies were prospectively followed for intrapartum complications (Table I), premature rupture of membranes, length of labor, and type of delivery, i.e, cesarean section or vaginal. Respiratory distress syndrome was diagnosed if all of the following criteria were met: (1) Silverman-Anderson retraction score was > 4 at 4 hours, (2) there was an audible expiratory grunt at 4 hours, (3) an increased ambient oxygen concentration was required for 24 hours to keep the Pao~> 40 mm Hg, and (4) there was radiologic evidence of RDS. Respiratory distress was diagnosed if the following criteria were met: (1) Silverman-Anderson retraction score was > 4 at 4 hours, (2) an increased ambient oxygen concentration was required for > 12 hours to keep the Pao2 > 40 mm Hg and, (3) a specific Clinical or radiologic diagnosis of lung disease could not be made, such as RDS, meconium aspiration, congenital heart disease, or pneumonia. The RD group
did include those infants with radiologic changes charac. teristic of transient tachypnea of the newborn infants~ This separation of infants with respiratory problems into two types is, of course, arbitrary; in some instances, RD is most likely a mild form of RDS. Total protein in amniotic fluid was determined by the technique of Lowry and associates 12and in the cord serum by refractometry. Albumin, al-AT, and a~-M concentrations in both amniotic fluid and cord sera were determined by radial diffusion in gel Using immunodiffusion plates obtained from Behring Diagnostics (Division of Hoechst Pharmaceuticals Co.): A recent comparison of six commercial kits used for a~-AT determination reported the least overall variability in results with Behring kits, indicated greater error when analyses w e r e made on specimens of high al-AT concentration, and suggested the need for uniform standards? 3 In order to minimize dayto-day and specimen-to-specimen procedural variations, the same set of four standards for each protein were used throughout the study; and care was taken to ensure equal filling of wells by employing an accurate microliter pipette. All tests were allowed to diffuse to equilibrium for 72 hours, and all reservoirs were utilized in each immunodiffusion chamber in order to ensure maximum reproducibility. Surface active material was assessed by the foam stability test as described by Clements and associates. 8 Volumes of amniotic fluid of 1.0, 0.75, 0.50, and 0.25 ml were pipetted into 14 • 100 mm tubes labeled 1/1, 1/1.3, 1/2, and 1/4, respectively. VoluMes of 0.25, 0.50, and 0.75 ml of 0.9% saline were pipetted into tubes Nos. 2, 3, and 4, respectively. One milliliter of 95% ethanol was added to each tube. The tubes were capped with clean rubber stoppers and shaken vigorously for 15 seconds by the clock and then placed vertically in a test-tube rack. Fifteen minutes later the air-liquid interface was examined in each tube for the presence of small, stable bubbles. Bright overhead light was used and the tubes viewed against a flat, black background. A tube was recorded as positive if it showed a complete ring of bubbles in the meniscus. If the tube labeled 1/1 was negative, the test was called negative. If a tube labeled 1/1 or 1/1.3 was positive, then the test was called intermediate; and a tube labeled 1/2 or 1/4 was positive, then the test was called positive. This test was performed immediately after obtaining the amniotic fluid. No sample of amniotic fluid contained meconium or blood. RESULTS Amniotic fluid alphal-antitrypsin was identified in the first trimester of gestation (Fig. 1) and appeared to rise to maximum levels by 25 to 35 weeks' gestation and to decrease toward term.
Volume 88 Number 1
A lpha t-antitrypsin in amniotic fluid
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Fig. 1. Amniotic fluid alphal-antitrypsin concentration (gm/dl) rises to maximum levels by 25 to 35 weeks' gestation and thereafter decreases toward term.
Fig. 2. The amniotic fluid protein concentration (gm/dl) is related to gestational age, rising in the first half of gestation to maximum levels by 25 to 35 weeks and thereafter decreasing toward term.
The amniotic fluid total protein concentration also appeared to be related to gestational age (Fig. 2), rising in the first half of gestation of maximum levels at 25 to 35 weeks and decreasing toward term. The data in Fig. 1 and 2 suggest a linear relationship between amniotic fluid alphal-antitrypsin and total protein, which on analysis was found to be true (r = 0.703, p < 0.001). Increasing concentrations of total protein were associated with increasing al-AT. Alpha2-macroglobulin was not detected in any of the amniotic fluid samples. If greater than 10% of the amniotic fluid protein represented contamination with maternal or fetal blood, a2-M would have been detected in the amniotic fluid. There was no correlation between the maternal serum concentration of al-AT or alphal-antitrypsin/total protein (a~-AT/TP) ratio and the amniotic fluid a~-AT or the alAT/TP. An attempt was made to determine whether the phenotype of the al-AT in amniotic fluids would identify the source as fetal or maternal. This was unsuccessful because there appeared to be binding of the al-AT to proteases that obscured the phenotypic patterns characteristic of the protein. At present there is no definitive information indicating whether amniotic fluid a~-AT is of maternal or fetal origin. The cord a1-AT concentration was linearly related to the cord total protein concentration (r = 0.571,
p < 0.001), the a,-AT generally rising with increasing total protein (Fig. 3). The cord al-AT is low in infants less than 25 weeks' gestation and reaches maximum concentration sometime between 25 weeks' gestation and term (Fig. 4). Infants with a history of intrapartum complications (Table I) had cord al-AT concentrations significantly lower than those infants without complications (p < 0.005, Fig. 4). This decrease in cord al-AT concentration with complications was not simply a function of a decreased cord total protein concentration, since there was also a significant decrease of cord alphal-antitrypsin/ total protein (p < 0.005). Thus, with intrapartum complications, the al-AT appears to be decreased more than total protein, even though the cord total protein is also significantly decreased (p < 0.025). Premature rupture of the membranes of duration _> 18 hours occurred in seven pregnancies of 26 to 35 weeks' gestation. Premature rutpure of the membranes was always associated with normal or high values of cord a~AT concentrations. The al-AT/TP ratio in particular was likely to be elevated. Thus with prolonged premature rupture of membranes, the a,-AT concentration was generally increased more than total protein concentration. Only two of the seven mothers with premature rupture of membranes had fever and evidence of amnionitis. The cord alpha~-macroglobulin concentration was linearly related to cord total protein (r = 0.739,
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Fig. 4. The cord alphas-antitrypsin is lowest in infants less than 26 weeks' gestation and appears to reach maximum concentrations .sometime between 26 weeks and term. Infants with a history of intrapartum complications had cord alphal-antitrypsin concentrations significantly lower than those infants without complications (p < 0.005).
p < 0.001) as shown in Fig. 5. The cord a~-M concentration like the as-AT was also significantly decreased when there was a history of intrapartum complications, but only in those infants less than 32 weeks' gestation. All infants with RDS were _< 36 weeks' gestation. The development of RDS was associated with a significant increase in amniotic fluid al-AT concentration (Table II). The amniotic fluid total protein concentrations were not significantly different in those fetuses that developed RDS when compared to those who did not (Table II). The study of the relationship of amniotic fluids as-AT in concentration to the foam stability test includes all amniotic fluids obtained within 24 hours of delivery in
pregnancies of gestational ages 26 to 42 weeks (Fig. 6). The al-AT does not have a consistent relationship with the FST. All infants with a negative FST had RDS regardless of the amniotic fluid al-AT concentration. However, RDS occurred in several infants with an intermediate or positive FST, and this was generally associated with an increase of as-AT above 0.05 gm/dl. There were only two instances in which the as-AT exceeded 0.05 gm/dl when the FST was intermediate, yet no RDS occurred. One of these infants was 37 weeks' gestation and had transient respiratory distress. The other was 31 weeks' gestation, under the tenth percentile for weight, and had no respiratory distress, but the mother had preeclampsia and had
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been on many drugs up to time of delivery. Fetuses who subsequently developed mild respiratory disturbances had normal levels of amniotic fluid alphal-antitrypsin. Seven of these nine fetuses had intrapartum complicafions. The relationship of intrapartum complications to the amniotic fluid a,-AT is difficult to assess, since the time course of complications is often uncertain and the kinetics of arrmiotic fluid a~-AT turnover are not known. The cord al-AT concentration did not appear to be related to the amniotic fluid surface active material as assessed by the foam stability test (Fig. 7). The respiratory distress syndrome occurred with a negative FST whether or not the cord a~-AT concentration was decreased. However, of the 23 infants with cord al-AT less than 0.2 gm/dl and with intermediate or positive FST, 19 infants had respiratory difficulties of varying severity. In contrast, there were 29 infants with a cord a~-AT concentration greater than 0.2 gm/dl and with an intermediate or positive FST, and only four infants had respiratory difficulties. All infants who died with RDS had histologic evidence of hyaline membranes. These infants had all received respirator therapy. DISCUSSION It is difficult to establish normal cord serum alpha,antitrypsin concentrations in preterm infants since many factors are operative during the intrapartum period, which may result in a decrease or an elevation of the a~AT. It appears that the cord serum a,-AT concentration is related to the total protein concentration. This suggests that a~-AT metabolism may be related to general protein metabolism in the fetus. Under certain circumstances,
Table II. The relationship of the respiratory distress syndrome in preterm infants _< 36 weeks' gestation to amniotic fluid* alphal-antitrypsin and total protein concentrations
] RDS [ NoRDS ] pvalue Number Alpha~-antitrypsin (mg/dl) Alpha~-antitrypsin/total protein Total proteins (gm/dl) Gestational age (wk)
20 65_+ 12t 127 _+19.3
47 27_+4.2 <0.005 68 _+7.9 <0.005
0.54_+0.09 0.41_+0.04 >0.05 32.3_+0.6 31.5_+0.7 >0.1
*Amniotic fluids collected within 24 hours o f delivery. "~Mean _4- 1 SE.
however, the production and catabolic rates of a~-AT may differ from other proteins including albumin. Perhaps this accounts for the greater drop in cord al-AT than total protein in pregnancies associated with intrapartum complications. Kotas and associates 1 have shown that infants who acquire intrauterine infections consistently have elevated levels of umbilical cord serum trypsin inhibitor capacity. Our study also has shown that rupture of membranes for over 18 hours before delivery was associated with high cord serum al-AT. Moreover, our observations suggest that intrapartum hypoxia and low Apgar scores are associated with depressed cord levels of a~-AT. The rise and fall of amniotic fluid alphal-antitrypsin and total protein during gestation is interesting. 1~ TM The cord alpha~-antitrypsin and protein concentrations also rise during the first two trimesters but do not decrease therafter. One possible explanation for the rise and fall of
92
Singer et al.
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The Journal of Pediatrics January 1976
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Fig. 6,The relationship of the amniofic fluid alpha]-antitrypsin to the amniotic fluid surface active material as assessed by the foam stability test. The two columns above the terms intermediate and positive refer to dilutions of amniotic fluid described under methods. There is a progressive increase in surfactant concentration as one moves from negative to positive at the highest dilution. The alphax-antitrypsin concentration does not appear to be related to the FST. Note that RDS occurred in the majority of infants with intermediate or positive FST when the alphalantitrypsin was elevated above 0.05 gm/dl.
protein in amniotic fluid would be that protein synthesis in the fetus increases during the first two trimesters when the capillaries are relatively permeable. As the capillaries become less permeable, t h e amniotic fluid alphaiantitrypsin and total protein decrease. Fierer and associates 6 showed that the decreased serum aI-AT in infants with RDS was probably not due to a failure of release o f a c A T from the liver. Our data in infants with respiratory disturbances are consistent with their findings, since the low cord al-AT was associated with high levels o f amniotic fluid a~-AT. This finding suggests losses from the blood rather than a decreased production o f a~-AT. This view is supported by the finding of Fierer and associates ' and Mathis and associates 5 who detected a~-AT as an integral part of the hyaline membranes o f infants who died with RDS.
Amniotic fluid and cord serum al-AT do not appear to be related to the amount of amniotic fluid surface active material. Infants with low amniotic fluid surface active material developed RDS regardless o f the concentrations of amniotic fluid or cord al-AT. On the other hand, those infants with intermediate or positive F S T who developed RDS had high levels of amniotic fluid al-AT and low levels of cord a~-AT. It is conceivable that these infants, in spite of apparent adequate prenatal lung surfactant, develop respiratory disturbances on the basis of pulmonary fluid transudation a n d / o r reduction of pulmonary surfactant incident to intrapartum complications, l'~ We are indebted to Dr. Jack Lieberman, City of Hope National Medical Center, Duarte, California, for attempting to compare the alphal-antitrypsin phenotypes of amniotic fluid with those of maternal serum. REFERENCES
1. Kotas RV, Fazen LE, and Moore TE: Umbilical cord serum trypsin inhibitor capacity and the idiopathic respiratory distress syndrome, J PEDIATR81:593, 1972. 2. Evans HE, Levi M, and Mandl I: Serum enzyme inhibitor concentrations in the respiratory distress syndrome, Am Rev Resp Dis 101:359, 1970. 3. Evans HE, Keller S, and Mandl I: Serum trypsin inhibitory capacity and the idiopathic respiratory distress syndrome, J PEDIATR81:588, 1972. 4. E1-Bardeesy MW, and Johnson AM: Serum proteinase inhibitors in infants with hyaline membrane disease, J PEDIATR81:579, 1972. 5. Mathis RK, Freier EF, Hunt CF, Krivit W, and Sharp HL:
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6.
7.
8.
9.
Alphal-antitrypsin in the respiratory distress syndrome, N Engl J Med 288:59, 1973. Fierer JA, Mandl I, and Evans HE: Alphal-antitrypsin in the lungs of newborn infants with respiratory distress syndrome, J PEDIATR85:698, 1974. Gtuck L Kulovich MV, Borer RC Jr, Brenner PH, Anderson GC, and Spellacy WN: The diagnosis of the respiratory distress syndrome (RDS) by amniocentesis, Am J Obstet Gynecol 109:440, 1971. Clements JA, Platzker ACG, Tierney DF, Hobel C J, Creasy RK, Margolis AJ, Thibeault DW, Tooley WH, and Oh W: Assessment of the risk of the respiratory distress syndrome by a rapid test for surfactant in amniotic fluid, N Engl J Med 286:10771 1972. Gitlin B, and Biasucci A: Development of,/G, -/A, "/M, Bic/ Bia, C'I esterase inhibitor, ceruloplasmin, transferrin, hemopexin, haptoglobin, fibrinogen, plasminogen, cq-antitrypsin, orosomucoid, B-lipoprotein, a2-macroglobulin, and prealbumin in the human conceptus, J Clin Invest 48:1433, 1969.
A lphafantitrypsin in amniotic fluid
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10. Guibaud S, Bonnet M, Thoulon JM, and Duman TM: Alphaa-antitrypsin in amniotic fluid, Obstet Gynecol 45:34, 1974. 11. Avery ME, Gatewood OE, and Brumley G: Transient tachypnea of the newborn, Am J Dis Child 111:380, 1966. 12. Lowry OH, Rosebrough NJ, Farr AL, and Randall PJ: Protein measurement with the Folin phenol reagent, J Biol Chem 198:265, 1951. 13. Seal LA, Carp DA, and George RB: Comparison of commercially available radial immunodiffusion kits for the determination of serum al-antitrypsin concentrations, Am Rev Resp Dis 111:97, 1975. 14. Queenan JT, Thompson W, Whitfield CR, and Shah SI: Amniotic fluid volumes in normal pregnancies, Am J Obstet Gynecol 114:34, 1972. 15. Gtuck L, Kulovich MV, Eidelman A, Cordero L, and Khazin AF: Biochemical development of surface activity in mammalian lung. IV. Pulmonary lecithin synthesis in the human fetus and newborn and etiology of the respiratory distress syndrome, Pediatr Res 6:81, 1972.
NEONATAL RichardE. Behrman,
Editor
Alphal-antitrypsin in amniotic fluid and cord blood of preterm infants with the respiratory distress syndrome Total protein, alphal-antitrypsin, and alpha2-macroglobulin were measured on amniotic fluid in 125 pregnancies between 11 and 42 weeks" gestation, and on the cord blood o f 66 newborn infants. Amniotic fluid surface active material was assessed by the foam stability test. Amniotic fluid alphal-antitrypsin is linearly and directly related to amniotic fluid total protein (r = O. 703, p < 0.001). The cord alphalantitrypsin is also linearly related to cord total protein, lntrapartum complication s are associated with a significant lowering o f the cord alphal-antitrypsin, lnfants with a negative foam stability test had R D S regardless of the amniotic fluid alpha,-antitrypsin concentration. However, R D S occurred in several infants with an intermediate or positive foam stability test; and this combination was generally associated with an increase o f amniotic fluid alphal-antitrypsin. The cord blood alphal-antitrypsin value did not appear to be related to amniotic fluid surface active material There were 23 infants with cord alphalantitrypsin of less than 0.2 gm/dl and with an intermediate or positive foam stability test; 19 o f them had respiratory difficulties o f varying severity. It is conceivable that infants', in spite o f apparent adequate prenatal lung surfactant, develop respiratory disturbances on the basis of pulmonary fluid and protein transudation and~or reduction or inhibition o f pulmonary surfactant incident to intrapartum complications.
Allan D. Singer, M.D.,* Donald W. Thibeault, M.D.,** Calvin J. Hobel, M.D., and Douglas C. Heiner, M.D., Ph.D., Torrance, Calif.
THE SERUM PROTEINASE INHIB!TORS, alphal-antitrypsin and alpha~,macroglobulin, have been shown to be decreased in the cord blood of infants who subsequently develop RDS. 1-~ Indeed, the outcome of infants with the respiratory distress syndrome may be predicted at birth from the umbilical cord serum levels of the serum trypsin inhibitor capacity. 1 Since the amniotic fluid surface active material is also highly predictive of the risk of a newborn infant to develop RDS, it would be of interest to explore the relationship of amniotic fluid surface material to cord From the Divisions of Perinatal Medicine and Immunology-A llergy, Departments o f Pediatrics and Obstetrics, Harbor General Hospital, UCLA School o f Medicine, *Supported in part by a grant from Hoechst Pharmaceuticals, Inc., Somerville, N. J. **Reprint address: Department of Pediatrics, Harbor General Hospital, 1000 West Carson St., Torrance, Calif. 90509.
and amniotic fluid alphacantitrypsin> 8 To our knowledge, a systematic study of amniotic fluid alphaxantitrypsin throughout gestations and of its relationship to amniotic fluid surface material has not been made> 9. ,o Abbreviations used al-AT: alphal-antitrypsin a2-M: alpha2-macroglobulin RD: respiratorydistress FST: foam stability test TP: total protein cord: umbilical cord It is the purpose of this communication to report studies regarding the concentrations of cord and amniotic fluid alphal-antitrypsinin normal and complicated pregnancies and the relationship of these concentrations to amniotic fluid surface active material. The Journal o f P E D I A T R IC S Vol. 88, No. 1, pp. 87-93
87
88
Singer et al.
The Journal of Pediatrics January 1976
Table I. Intrapartum factors* considered to be detrimental to the fetus in 66 high-risk pregnancies
I No. Five minute apgar score --<6"~ Intrapartum bleeding Breech delivery -<33 weeks' gestation Twins delivered vaginally --<33weeks Maternal hypotension Seizures (eclampsia) Type II deceleration fetal ECG pattern Diabetic ketoacidosis Prolonged second stage labor >2 hours
13 3 3 2 5 1 4 1 1
*Some subjects had more than one factor. t A low apgar score is a postnatal factor strongly suggestive o f an ill fetus.
MATERIAL
AND METHODS
Total protein, albumin, al-AT, a~-M, and surface active material were measured in amniotic fluids of 125 pregnancies of 11 to 42 weeks' gestation. Amniotic fluid was obtained by transabdominal amniocentesis or needle aspiration through the bulging forewaters. Sixty-seven samples of amniotic fluid were obtained within 24 hours of delivery; 15 were prior to therapeutic abortion, and the remaining 43 samples were obtained 24 hours or more prior to delivery. In 44 pregnancies maternal venous blood was obtained at the time of amniotic fluid collection for a~-AT and a~-M measurement. Total protein, albumin, and a1-AT in the cord serum were measured in 66 infants, but a2-M was measured only in 53. The gestational ages of the infants ranged from 17 to 40 weeks. In 35 of these pregnancies, amniotic fluid was also obtained within 24 hours of delivery and studied as above. Sixty-six pregnancies were prospectively followed for intrapartum complications (Table I), premature rupture of membranes, length of labor, and type of delivery, i.e, cesarean section or vaginal. Respiratory distress syndrome was diagnosed if all of the following criteria were met: (1) Silverman-Anderson retraction score was > 4 at 4 hours, (2) there was an audible expiratory grunt at 4 hours, (3) an increased ambient oxygen concentration was required for 24 hours to keep the Pao~> 40 mm Hg, and (4) there was radiologic evidence of RDS. Respiratory distress was diagnosed if the following criteria were met: (1) Silverman-Anderson retraction score was > 4 at 4 hours, (2) an increased ambient oxygen concentration was required for > 12 hours to keep the Pao2 > 40 mm Hg and, (3) a specific Clinical or radiologic diagnosis of lung disease could not be made, such as RDS, meconium aspiration, congenital heart disease, or pneumonia. The RD group
did include those infants with radiologic changes charac. teristic of transient tachypnea of the newborn infants~ This separation of infants with respiratory problems into two types is, of course, arbitrary; in some instances, RD is most likely a mild form of RDS. Total protein in amniotic fluid was determined by the technique of Lowry and associates 12and in the cord serum by refractometry. Albumin, al-AT, and a~-M concentrations in both amniotic fluid and cord sera were determined by radial diffusion in gel Using immunodiffusion plates obtained from Behring Diagnostics (Division of Hoechst Pharmaceuticals Co.): A recent comparison of six commercial kits used for a~-AT determination reported the least overall variability in results with Behring kits, indicated greater error when analyses w e r e made on specimens of high al-AT concentration, and suggested the need for uniform standards? 3 In order to minimize dayto-day and specimen-to-specimen procedural variations, the same set of four standards for each protein were used throughout the study; and care was taken to ensure equal filling of wells by employing an accurate microliter pipette. All tests were allowed to diffuse to equilibrium for 72 hours, and all reservoirs were utilized in each immunodiffusion chamber in order to ensure maximum reproducibility. Surface active material was assessed by the foam stability test as described by Clements and associates. 8 Volumes of amniotic fluid of 1.0, 0.75, 0.50, and 0.25 ml were pipetted into 14 • 100 mm tubes labeled 1/1, 1/1.3, 1/2, and 1/4, respectively. VoluMes of 0.25, 0.50, and 0.75 ml of 0.9% saline were pipetted into tubes Nos. 2, 3, and 4, respectively. One milliliter of 95% ethanol was added to each tube. The tubes were capped with clean rubber stoppers and shaken vigorously for 15 seconds by the clock and then placed vertically in a test-tube rack. Fifteen minutes later the air-liquid interface was examined in each tube for the presence of small, stable bubbles. Bright overhead light was used and the tubes viewed against a flat, black background. A tube was recorded as positive if it showed a complete ring of bubbles in the meniscus. If the tube labeled 1/1 was negative, the test was called negative. If a tube labeled 1/1 or 1/1.3 was positive, then the test was called intermediate; and a tube labeled 1/2 or 1/4 was positive, then the test was called positive. This test was performed immediately after obtaining the amniotic fluid. No sample of amniotic fluid contained meconium or blood. RESULTS Amniotic fluid alphal-antitrypsin was identified in the first trimester of gestation (Fig. 1) and appeared to rise to maximum levels by 25 to 35 weeks' gestation and to decrease toward term.
Volume 88 Number 1
A lpha t-antitrypsin in amniotic fluid
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Fig. 1. Amniotic fluid alphal-antitrypsin concentration (gm/dl) rises to maximum levels by 25 to 35 weeks' gestation and thereafter decreases toward term.
Fig. 2. The amniotic fluid protein concentration (gm/dl) is related to gestational age, rising in the first half of gestation to maximum levels by 25 to 35 weeks and thereafter decreasing toward term.
The amniotic fluid total protein concentration also appeared to be related to gestational age (Fig. 2), rising in the first half of gestation of maximum levels at 25 to 35 weeks and decreasing toward term. The data in Fig. 1 and 2 suggest a linear relationship between amniotic fluid alphal-antitrypsin and total protein, which on analysis was found to be true (r = 0.703, p < 0.001). Increasing concentrations of total protein were associated with increasing al-AT. Alpha2-macroglobulin was not detected in any of the amniotic fluid samples. If greater than 10% of the amniotic fluid protein represented contamination with maternal or fetal blood, a2-M would have been detected in the amniotic fluid. There was no correlation between the maternal serum concentration of al-AT or alphal-antitrypsin/total protein (a~-AT/TP) ratio and the amniotic fluid a~-AT or the alAT/TP. An attempt was made to determine whether the phenotype of the al-AT in amniotic fluids would identify the source as fetal or maternal. This was unsuccessful because there appeared to be binding of the al-AT to proteases that obscured the phenotypic patterns characteristic of the protein. At present there is no definitive information indicating whether amniotic fluid a~-AT is of maternal or fetal origin. The cord a1-AT concentration was linearly related to the cord total protein concentration (r = 0.571,
p < 0.001), the a,-AT generally rising with increasing total protein (Fig. 3). The cord al-AT is low in infants less than 25 weeks' gestation and reaches maximum concentration sometime between 25 weeks' gestation and term (Fig. 4). Infants with a history of intrapartum complications (Table I) had cord al-AT concentrations significantly lower than those infants without complications (p < 0.005, Fig. 4). This decrease in cord al-AT concentration with complications was not simply a function of a decreased cord total protein concentration, since there was also a significant decrease of cord alphal-antitrypsin/ total protein (p < 0.005). Thus, with intrapartum complications, the al-AT appears to be decreased more than total protein, even though the cord total protein is also significantly decreased (p < 0.025). Premature rupture of the membranes of duration _> 18 hours occurred in seven pregnancies of 26 to 35 weeks' gestation. Premature rutpure of the membranes was always associated with normal or high values of cord a~AT concentrations. The al-AT/TP ratio in particular was likely to be elevated. Thus with prolonged premature rupture of membranes, the a,-AT concentration was generally increased more than total protein concentration. Only two of the seven mothers with premature rupture of membranes had fever and evidence of amnionitis. The cord alpha~-macroglobulin concentration was linearly related to cord total protein (r = 0.739,
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Fig. 4. The cord alphas-antitrypsin is lowest in infants less than 26 weeks' gestation and appears to reach maximum concentrations .sometime between 26 weeks and term. Infants with a history of intrapartum complications had cord alphal-antitrypsin concentrations significantly lower than those infants without complications (p < 0.005).
p < 0.001) as shown in Fig. 5. The cord a~-M concentration like the as-AT was also significantly decreased when there was a history of intrapartum complications, but only in those infants less than 32 weeks' gestation. All infants with RDS were _< 36 weeks' gestation. The development of RDS was associated with a significant increase in amniotic fluid al-AT concentration (Table II). The amniotic fluid total protein concentrations were not significantly different in those fetuses that developed RDS when compared to those who did not (Table II). The study of the relationship of amniotic fluids as-AT in concentration to the foam stability test includes all amniotic fluids obtained within 24 hours of delivery in
pregnancies of gestational ages 26 to 42 weeks (Fig. 6). The al-AT does not have a consistent relationship with the FST. All infants with a negative FST had RDS regardless of the amniotic fluid al-AT concentration. However, RDS occurred in several infants with an intermediate or positive FST, and this was generally associated with an increase of as-AT above 0.05 gm/dl. There were only two instances in which the as-AT exceeded 0.05 gm/dl when the FST was intermediate, yet no RDS occurred. One of these infants was 37 weeks' gestation and had transient respiratory distress. The other was 31 weeks' gestation, under the tenth percentile for weight, and had no respiratory distress, but the mother had preeclampsia and had
Volume 88 Number 1
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been on many drugs up to time of delivery. Fetuses who subsequently developed mild respiratory disturbances had normal levels of amniotic fluid alphal-antitrypsin. Seven of these nine fetuses had intrapartum complicafions. The relationship of intrapartum complications to the amniotic fluid a,-AT is difficult to assess, since the time course of complications is often uncertain and the kinetics of arrmiotic fluid a~-AT turnover are not known. The cord al-AT concentration did not appear to be related to the amniotic fluid surface active material as assessed by the foam stability test (Fig. 7). The respiratory distress syndrome occurred with a negative FST whether or not the cord a~-AT concentration was decreased. However, of the 23 infants with cord al-AT less than 0.2 gm/dl and with intermediate or positive FST, 19 infants had respiratory difficulties of varying severity. In contrast, there were 29 infants with a cord a~-AT concentration greater than 0.2 gm/dl and with an intermediate or positive FST, and only four infants had respiratory difficulties. All infants who died with RDS had histologic evidence of hyaline membranes. These infants had all received respirator therapy. DISCUSSION It is difficult to establish normal cord serum alpha,antitrypsin concentrations in preterm infants since many factors are operative during the intrapartum period, which may result in a decrease or an elevation of the a~AT. It appears that the cord serum a,-AT concentration is related to the total protein concentration. This suggests that a~-AT metabolism may be related to general protein metabolism in the fetus. Under certain circumstances,
Table II. The relationship of the respiratory distress syndrome in preterm infants _< 36 weeks' gestation to amniotic fluid* alphal-antitrypsin and total protein concentrations
] RDS [ NoRDS ] pvalue Number Alpha~-antitrypsin (mg/dl) Alpha~-antitrypsin/total protein Total proteins (gm/dl) Gestational age (wk)
20 65_+ 12t 127 _+19.3
47 27_+4.2 <0.005 68 _+7.9 <0.005
0.54_+0.09 0.41_+0.04 >0.05 32.3_+0.6 31.5_+0.7 >0.1
*Amniotic fluids collected within 24 hours o f delivery. "~Mean _4- 1 SE.
however, the production and catabolic rates of a~-AT may differ from other proteins including albumin. Perhaps this accounts for the greater drop in cord al-AT than total protein in pregnancies associated with intrapartum complications. Kotas and associates 1 have shown that infants who acquire intrauterine infections consistently have elevated levels of umbilical cord serum trypsin inhibitor capacity. Our study also has shown that rupture of membranes for over 18 hours before delivery was associated with high cord serum al-AT. Moreover, our observations suggest that intrapartum hypoxia and low Apgar scores are associated with depressed cord levels of a~-AT. The rise and fall of amniotic fluid alphal-antitrypsin and total protein during gestation is interesting. 1~ TM The cord alpha~-antitrypsin and protein concentrations also rise during the first two trimesters but do not decrease therafter. One possible explanation for the rise and fall of
92
Singer et al.
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The Journal of Pediatrics January 1976
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Fig. 6,The relationship of the amniofic fluid alpha]-antitrypsin to the amniotic fluid surface active material as assessed by the foam stability test. The two columns above the terms intermediate and positive refer to dilutions of amniotic fluid described under methods. There is a progressive increase in surfactant concentration as one moves from negative to positive at the highest dilution. The alphax-antitrypsin concentration does not appear to be related to the FST. Note that RDS occurred in the majority of infants with intermediate or positive FST when the alphalantitrypsin was elevated above 0.05 gm/dl.
protein in amniotic fluid would be that protein synthesis in the fetus increases during the first two trimesters when the capillaries are relatively permeable. As the capillaries become less permeable, t h e amniotic fluid alphaiantitrypsin and total protein decrease. Fierer and associates 6 showed that the decreased serum aI-AT in infants with RDS was probably not due to a failure of release o f a c A T from the liver. Our data in infants with respiratory disturbances are consistent with their findings, since the low cord al-AT was associated with high levels o f amniotic fluid a~-AT. This finding suggests losses from the blood rather than a decreased production o f a~-AT. This view is supported by the finding of Fierer and associates ' and Mathis and associates 5 who detected a~-AT as an integral part of the hyaline membranes o f infants who died with RDS.
Amniotic fluid and cord serum al-AT do not appear to be related to the amount of amniotic fluid surface active material. Infants with low amniotic fluid surface active material developed RDS regardless o f the concentrations of amniotic fluid or cord al-AT. On the other hand, those infants with intermediate or positive F S T who developed RDS had high levels of amniotic fluid al-AT and low levels of cord a~-AT. It is conceivable that these infants, in spite of apparent adequate prenatal lung surfactant, develop respiratory disturbances on the basis of pulmonary fluid transudation a n d / o r reduction of pulmonary surfactant incident to intrapartum complications, l'~ We are indebted to Dr. Jack Lieberman, City of Hope National Medical Center, Duarte, California, for attempting to compare the alphal-antitrypsin phenotypes of amniotic fluid with those of maternal serum. REFERENCES
1. Kotas RV, Fazen LE, and Moore TE: Umbilical cord serum trypsin inhibitor capacity and the idiopathic respiratory distress syndrome, J PEDIATR81:593, 1972. 2. Evans HE, Levi M, and Mandl I: Serum enzyme inhibitor concentrations in the respiratory distress syndrome, Am Rev Resp Dis 101:359, 1970. 3. Evans HE, Keller S, and Mandl I: Serum trypsin inhibitory capacity and the idiopathic respiratory distress syndrome, J PEDIATR81:588, 1972. 4. E1-Bardeesy MW, and Johnson AM: Serum proteinase inhibitors in infants with hyaline membrane disease, J PEDIATR81:579, 1972. 5. Mathis RK, Freier EF, Hunt CF, Krivit W, and Sharp HL:
Volume 88 Number 1
6.
7.
8.
9.
Alphal-antitrypsin in the respiratory distress syndrome, N Engl J Med 288:59, 1973. Fierer JA, Mandl I, and Evans HE: Alphal-antitrypsin in the lungs of newborn infants with respiratory distress syndrome, J PEDIATR85:698, 1974. Gtuck L Kulovich MV, Borer RC Jr, Brenner PH, Anderson GC, and Spellacy WN: The diagnosis of the respiratory distress syndrome (RDS) by amniocentesis, Am J Obstet Gynecol 109:440, 1971. Clements JA, Platzker ACG, Tierney DF, Hobel C J, Creasy RK, Margolis AJ, Thibeault DW, Tooley WH, and Oh W: Assessment of the risk of the respiratory distress syndrome by a rapid test for surfactant in amniotic fluid, N Engl J Med 286:10771 1972. Gitlin B, and Biasucci A: Development of,/G, -/A, "/M, Bic/ Bia, C'I esterase inhibitor, ceruloplasmin, transferrin, hemopexin, haptoglobin, fibrinogen, plasminogen, cq-antitrypsin, orosomucoid, B-lipoprotein, a2-macroglobulin, and prealbumin in the human conceptus, J Clin Invest 48:1433, 1969.
A lphafantitrypsin in amniotic fluid
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10. Guibaud S, Bonnet M, Thoulon JM, and Duman TM: Alphaa-antitrypsin in amniotic fluid, Obstet Gynecol 45:34, 1974. 11. Avery ME, Gatewood OE, and Brumley G: Transient tachypnea of the newborn, Am J Dis Child 111:380, 1966. 12. Lowry OH, Rosebrough NJ, Farr AL, and Randall PJ: Protein measurement with the Folin phenol reagent, J Biol Chem 198:265, 1951. 13. Seal LA, Carp DA, and George RB: Comparison of commercially available radial immunodiffusion kits for the determination of serum al-antitrypsin concentrations, Am Rev Resp Dis 111:97, 1975. 14. Queenan JT, Thompson W, Whitfield CR, and Shah SI: Amniotic fluid volumes in normal pregnancies, Am J Obstet Gynecol 114:34, 1972. 15. Gtuck L, Kulovich MV, Eidelman A, Cordero L, and Khazin AF: Biochemical development of surface activity in mammalian lung. IV. Pulmonary lecithin synthesis in the human fetus and newborn and etiology of the respiratory distress syndrome, Pediatr Res 6:81, 1972.