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Serum trypsin inhibitory capacity and the idiopathic respiratory distress syndrome
588
September, 1972 T h e J o u r n a l of P E D I A T R I C S
Serum trypsin inl 'bitory capacity and the idiopathic respiratory distress syndrome Alpha-1 antitrypsin, alpha-2 macroglobuIin, and total trypsin inhibitory capacity levels were significantly lower in the umbilical cord serum o[ newborn in[ants with idiopathic respiratory distress syndrome ( I R D S ) than in weight-matched control subjects. Levels were unrelated to gestational age or sex. Sequential serum samples were drawn in 17 in[ants with I R D S ; there was a tendency [or levels o[ total trypsin inhibitory capacity to increase in 6 o[ 9 survivors. In contrast, 7 o[ the 8 in[ants who died had persistently low levels and some had [urther decreases in concentration. In amniotie fluid obtained at 12 to 22 weeks" gestation in cases not related to IRDS, alpha-1 antitrypsin and total trypsin inhibitory capacity could usually be detected but alpha-2 macroglobulin was not. Serum enzyme inhibitor levels may be o[ diagnostic significance in IRDS.
Hugh E. Evans, M.D., Stephen Keller, Ph.D., and Ines Mandl, Ph.D., N e w Y o r k , N . Y.
E T I o L 0 0 Y and pathogenesis of the idiopathic respiratory distress syndrome (IRDS) are unknown, although many hypotheses have been advanced including surfactant deficiency, pulmonary hypoperfusion, and fibrinolyric enzyme deficiency? We have previously reported decreased serum levels of inhibitors of elastase and trypsin in cord sera of newborn infants with IRDS. 2 Further studies have confirmed the initial From the Department o[ Pediatrics, Harlem Hospital Center, and the Department of Obstetrics and Gynecdogy, Francis Delafield Hospital, Columbia University College o[ Physicians & Surgeong. Supported in part by grants from the National Foundation, The New York Tuberculosis and Respiratory Disease Association, and National Heart and Lung Institute S C O R Grant H L 14218. Reprint address: Hugh IE. Evans, M.D., Department of Pediatrics, Harlem Hospital Center, New York, N. Y. 10037.
Vol. 8l, No. 3, pp. 588-592
observation concerning trypsin inhibitor, have revealed decreased levels of alpha-2 macroglobulin, and have shown a tendency for inhibitor levels to increase during the first two days of life among survivors of IRDS. Normal values were found at term in the mothers of newborn infants with IRDS.
MATERIALS AND M E T H O D S Alpha-1 antitrypsin, alpha-2 macroglobulin, and total trypsin inhibitory capacity were determined in the serum of three groups of newborn infants and several of the mothers of those with I R D S and in amniotic fluid specimens unrelated to IRDS. A group of 37 newborn infants, 25 of them males, had clinical, biochemical, and, in 20 of the 25 fatal cases, histopathologic evidence of IRDS. Three infants were delivered by cesarean section. Birth weights ranged from
Volume 81 Number 3
Serum trypsin inhibitory capacity and R D S
589
Table I. Level of serum enzyme inhibitor components in newborn infants with idiopathic respiratory distress syndrome ( I R D S ) , their mothers, and control subjects
Group Infants with IRDS ~ Low-birth-weight infants without IRDS ~ Full-term infants 'k Mothers of infants with IRDS Infants with IRDS versus low-birthweight infants without IRDS Infants with IRDS versus full-term infants
Alpha-I antitrypsin (rag./lO0 ml.) No. tested Mean Range 19 104 51-200
Trypsin inhibitory capacity Alpha-2 raacrogIobulin (rag. trypsin inhibited/ml. (mg./lO0 ml.) serum) No. ~ No. tested Mean Range tested Mean, Range 0.48 0.00-1.32 238 65- 837 29 20
26 21
193 228
96-300 156-432
20 28
422 380
195-1,260 165- 950
35 23
0.94 1.22
0.16-1.77 0.61-1.65
16
288
152-448
11
388
175- 675
17
1.09
0.85-1.48
p < 0.01
p < 0.01
p < 0.05
p < 0.0.1
p < 0.0.1
p < 0.01
*UmbJJical cord sera only.
600 to 2,840 Gin. with a m e a n of 1,254 Gm. A control group of newborn infants without I R D S , 25 of them males, in a comparable weight range (482 to 2,200 Gin. with a mean of 1,230 Gin.) were studied in the same way. Two of them were delivered by cesarean section. Five of this group, all immature, died. I n addition, a group of 28 healthy full-term infants, 14 of them males, was studied, and blood was also drawn from 18 mothers of neonates with I R D S , one of whom was diabetic. Amniotic fluid was obtained by amniocentesis at 12 to 22 weeks' gestation as an initial step in 30 w o m e n in w h o m salineinduced abortion of ~oresumably normal fetuses was to be performed. I n three additional cases erythroblastosis fetalis or other conditions were under consideration. Serum trypsin inhibitory capacity was determined by slight modification of a method described in detail by Erlanger and associates. s T h e procedure involves incubation of test serum, buffer, enzyme, and the chromogenic substrate benzoyl arginine p-nitroanilide. Results of colorimetric assays of the p-nitro-aniline released were compared with values obtained with corresponding enzyme concentrations in the absence of serum.
Alpha-1 antitrypsin and alpha-2 macroglobulin were measured by agar gel immunodiffusion on commercially available "Partigen" plates (Behringwerke).4 A m o n g the newborn infants, these determinations were carried out on umbilical cord sera or sera obtained within a few hours of life. T h e latter specimens were excluded from Table I. RESULTS
As shown in Table I, the concentrations of alpha-1 antitrypsin and alpha-2 macroglobulin and the total serum trypsin inhibitory capacity were significantly reduced in the infants with I R D S compared with lowbirth-weight infants without I R D S and full-term infants. Serum levels of enzyme inhibitors at the time of delivery a m o n g the mothers of infants with I R D S were comparable to those of low-birth-weight infants without I R D S and full-term infants. No correlation was found between the level of enzyme inhibitors (alpha-1 antitrypsin, alpha-2 macroglobulin, or total serum trypsin inhibitory capacity) and birth weight, intrauterine growth, or sex. I n 17 infants with I R D S , two or more serum samples were obtained over a period of several hours or days. Eight of these infants
590
The Journal of Pediatrics September 1972
Evans, Keller, and Mandl
].6--
E 1.4--
-
--
@=Case 1 e:Case 2 g=Case 3
@:Case 4 e~=Case5 A:Case 6
k=Case 7 r'l:Case 8
1.2--
04
0
1
2
3
4
5 6 Hours
7
8
9
10 // 32
Fig. 1A. Serial total serum trypsin inhibitor determination with respiratory distress syndrome-fatal cases.
1.6 - -
@=Case 1 @:Case2 g:Case3
-i
1.4-
N
@=Case4 e~Case 5 A'Case 6
A=Case7 El:Case 8 II--Case 9 . . . . . . . . ~ . . . . ~
A
1.0'
-r-
_z 0.8 _0.6
~ 0.4 ~
I~1 0
2
4
i [ I i I r i i i I ] i.I 6
8 16 24 26 28 30 32 38 Hours
52
72 80 96"3
I I 5 6 Days
Fig. lB. Serial total serum trypsin inhibitor determination with respiratory distress syndrome-survivors.
died (mean weight, 1,290 Gm.), and seven of these had persistently low levels, and in some still further decreases, of total serum trypsin inhibitory capacity. Only in one fatal case (Case 4) did the infant's level rise. Of the nine survivors (mean birth weight 1,422 Gin.), six had increases in serum levels of trypsin inhibitor, two had normal levels throughout the clinical course (Cases 6 and
7), and in only one of the survivors (Case 9) did the levels remain persistently low (Figs. 1A and 1B). Alpha-1 antitrypsin was measurable in 28 of 33 samples of amniotic fluid, and total trypsin inhibitory capacity was detectable in each of the 20 samples tested. In contrast, alpha-2 macroglobulin was measurable in amniotic fluid in only 1 of 29 samples. The lower limit of "detectability is
Volume 81 Number 3
Serum trypsin inhibitory capacity and RDS
about 10 mg. per 100 ml. for alpha-1 antitrypsin and about 20 rag. per 100 ml. for alpha-2 macroglobulin. DISCUSSION The data suggest that decreased levels of alpha-1 antitrypsin, alpha-2 macroglobulin, and total serum trypsin inhibitory capacity may be of diagnostic significance in IRDS. Levels of serum alpha-1 antitrypsin and total serum trypsin inhibitory capacity found in neonates with I R D S correspond to those reported in adults with heterozygous and, in some cases, homozygous alpha-1 antitrypsin deficiency. Levels found in low-birth-weight infants without IRDS, full-term infants, and mothers of neonates with I R D S correspond to values reported in normal adults. 5"7 Levels in the low-birth-weight infants without I R D S were not significantly different from those observed in the full-term infants. Alpha-2 macroglobulin levels observed in our laboratory were higher among full-term newborn infants, low-birth-weight control infants, and mothers than those recently reported by Howie and associates s in adult women. Hyvarinan and associates 9 found no difference in either serum alpha-1 antitrypsin or alpha-2 macroglobulin concentrations between low-birth-weight infants with or without IRDS. Normal serum levels of alpha-2 macroglobulin and inter-alpha inhibitor were reported among adults with alpha-1 antitrypsin deficiency? ~ Persistent low serum trypsin inhibitory capacity in newborn .infants with I R D S may be of prognostic significance. The observations reported are not completely controlled, since the patients with persistently low levels died in 10 hours or less, whereas survivors were studied for several days. Further studies are needed before definite conclusions are warranted. However, it is of interest that levels can change with time during the first few days of life and that increases appear to be associated with improved prognosis, and vice versa. The data confirm our previous findings" and those of others 9 that immaturity, per se, does not seem to predispose to serum trypsin
59 1
inhibitor deficiency. Familial predisposition to I R D S has been previously reported, al, 12 but normal levels of total serum trypsin inhibitory capacity, alpha-1 antitrypsin, and alpha-2 macroglobulin observed at term in mothers of newborn infants with I R D S are not indicative of a genetic determinant of inhibitor deficiency. Studies of phenotypes of the probands and of trypsin inhibitor levels in siblings and fathers and in mothers 6 weeks postpartum or later have not been completed. Normal values in maternal sera at term do not rule out possible heterozygous deficiency, since titers are known to be elevated at that time? s The presence of alpha-1 antitrypsin in amniotic fluid suggests that fetal synthesis, or passive acquisition, occurs early in gestation. This may allow early screening for the deficiency in mothers at high risk. The apparent absence of alpha-2-macroglobulin may be due to relatively delayed synthesis or passive transfer. The authors wish to thank Mrs. Yvonne Hosannah, Miss Mabel Wong, and Mr. Solomon O. Akpata for carrying out the serum enzyme inhibitor tests. Dr. Michael Levi performed the amniocenteses. REFERENCES
1. Klaus, M. It.: Respiratory function and pulmonary disease in the newborn, in Barnett, H., editor: Pediatrics, ed. 14, New York, 1969, Appleton-Century-Crofts, Inc., pp. 1422-1435. 2. Evans, H. E., Levi, M., and Mandl, I.: Serum enzyme inhibitor concentrations in the respiratory distress syndrome, Am. Rev. Resp. Dis. 101: 359, 1970. 3. Erlanger, B. F., Kokowsky, N., and Cohen, W.: The preparation and properties of two new chromogenic substrates of trypsin, Arch. Biochem. Biophys. 95: 271, 1961. 4. Mancinl, G., Carbonara, A. O., and Hereroans, J. F.: Immunochemical quantltation of antigens by single radial immunodiffusion, Immunochemistry 2: 235, 1965. 5. Lieberman, J.: Heterozygous and homozygous alpha-1 antitrypsin deficiency in patients with pulmonary emphysema, N. Engl. J. Med. 281: 270, 1969. 6. Maklno, S., Chosy, L., Valdivia, E., and Reed, C. E.: Emphysema with alpha-1 antitrypsin masquerading as asthma, J. Allergy 46: 40, 1970.
59 2
Evans, Keller, and Mandl
7. Kueppers, F.: Immunologic assay of alpha-1 antitrypsin in deficient subjects and their families, Humangenetik 5: 54, 1967. 8. Howie, P. W., Mallinson, A. C., Prentice, C. R. M., Home, C. H. W., and McNicol, G. P.: Effect of combined estrogen-progestogen oral contraceptives, estrogen and progestogen on antiplasmin and antithrombin activity, Lancet 2: 1329, 1970. 9. Hyvarinan, M., Graven, S. N., and Stiehm, E. R.: Development of fibrinolytic proteins in health and disease, Proc. Soc. Pediatr. Res., 1970, p. 86. 10. Ganrot, P. O., Laurell, C. B., and Eriksson,
The ]ournal of Pediatrics September 1972
S.: Obstructive lung disease and trypsin inhibitors in alpha-1 antitrypsin deficiency, Scand. J. Clin. Lab. Invest. 19: 205, 1967. 11. Graven, S., and Misenheimer, H.: Respiratory distress syndrome and the high risk mother, Am. J. Dis. Child. 109: 489, 1965. 12. Graven, S., Opitz, J. M., and Harrison, M.: The respiratory distress syndrome, Am. J. Obstet. Gynecol. 96: 909, 1966. 13. Sciarra, J. J., Chao, S., and Mandl, I.: Quantitative assay of elastase inhibitor in serum during pregnancy, Am. J. Obstet. Gynecol. 86: 753, 1963.
September, 1972 T h e J o u r n a l of P E D I A T R I C S
Serum trypsin inl 'bitory capacity and the idiopathic respiratory distress syndrome Alpha-1 antitrypsin, alpha-2 macroglobuIin, and total trypsin inhibitory capacity levels were significantly lower in the umbilical cord serum o[ newborn in[ants with idiopathic respiratory distress syndrome ( I R D S ) than in weight-matched control subjects. Levels were unrelated to gestational age or sex. Sequential serum samples were drawn in 17 in[ants with I R D S ; there was a tendency [or levels o[ total trypsin inhibitory capacity to increase in 6 o[ 9 survivors. In contrast, 7 o[ the 8 in[ants who died had persistently low levels and some had [urther decreases in concentration. In amniotie fluid obtained at 12 to 22 weeks" gestation in cases not related to IRDS, alpha-1 antitrypsin and total trypsin inhibitory capacity could usually be detected but alpha-2 macroglobulin was not. Serum enzyme inhibitor levels may be o[ diagnostic significance in IRDS.
Hugh E. Evans, M.D., Stephen Keller, Ph.D., and Ines Mandl, Ph.D., N e w Y o r k , N . Y.
E T I o L 0 0 Y and pathogenesis of the idiopathic respiratory distress syndrome (IRDS) are unknown, although many hypotheses have been advanced including surfactant deficiency, pulmonary hypoperfusion, and fibrinolyric enzyme deficiency? We have previously reported decreased serum levels of inhibitors of elastase and trypsin in cord sera of newborn infants with IRDS. 2 Further studies have confirmed the initial From the Department o[ Pediatrics, Harlem Hospital Center, and the Department of Obstetrics and Gynecdogy, Francis Delafield Hospital, Columbia University College o[ Physicians & Surgeong. Supported in part by grants from the National Foundation, The New York Tuberculosis and Respiratory Disease Association, and National Heart and Lung Institute S C O R Grant H L 14218. Reprint address: Hugh IE. Evans, M.D., Department of Pediatrics, Harlem Hospital Center, New York, N. Y. 10037.
Vol. 8l, No. 3, pp. 588-592
observation concerning trypsin inhibitor, have revealed decreased levels of alpha-2 macroglobulin, and have shown a tendency for inhibitor levels to increase during the first two days of life among survivors of IRDS. Normal values were found at term in the mothers of newborn infants with IRDS.
MATERIALS AND M E T H O D S Alpha-1 antitrypsin, alpha-2 macroglobulin, and total trypsin inhibitory capacity were determined in the serum of three groups of newborn infants and several of the mothers of those with I R D S and in amniotic fluid specimens unrelated to IRDS. A group of 37 newborn infants, 25 of them males, had clinical, biochemical, and, in 20 of the 25 fatal cases, histopathologic evidence of IRDS. Three infants were delivered by cesarean section. Birth weights ranged from
Volume 81 Number 3
Serum trypsin inhibitory capacity and R D S
589
Table I. Level of serum enzyme inhibitor components in newborn infants with idiopathic respiratory distress syndrome ( I R D S ) , their mothers, and control subjects
Group Infants with IRDS ~ Low-birth-weight infants without IRDS ~ Full-term infants 'k Mothers of infants with IRDS Infants with IRDS versus low-birthweight infants without IRDS Infants with IRDS versus full-term infants
Alpha-I antitrypsin (rag./lO0 ml.) No. tested Mean Range 19 104 51-200
Trypsin inhibitory capacity Alpha-2 raacrogIobulin (rag. trypsin inhibited/ml. (mg./lO0 ml.) serum) No. ~ No. tested Mean Range tested Mean, Range 0.48 0.00-1.32 238 65- 837 29 20
26 21
193 228
96-300 156-432
20 28
422 380
195-1,260 165- 950
35 23
0.94 1.22
0.16-1.77 0.61-1.65
16
288
152-448
11
388
175- 675
17
1.09
0.85-1.48
p < 0.01
p < 0.01
p < 0.05
p < 0.0.1
p < 0.0.1
p < 0.01
*UmbJJical cord sera only.
600 to 2,840 Gin. with a m e a n of 1,254 Gm. A control group of newborn infants without I R D S , 25 of them males, in a comparable weight range (482 to 2,200 Gin. with a mean of 1,230 Gin.) were studied in the same way. Two of them were delivered by cesarean section. Five of this group, all immature, died. I n addition, a group of 28 healthy full-term infants, 14 of them males, was studied, and blood was also drawn from 18 mothers of neonates with I R D S , one of whom was diabetic. Amniotic fluid was obtained by amniocentesis at 12 to 22 weeks' gestation as an initial step in 30 w o m e n in w h o m salineinduced abortion of ~oresumably normal fetuses was to be performed. I n three additional cases erythroblastosis fetalis or other conditions were under consideration. Serum trypsin inhibitory capacity was determined by slight modification of a method described in detail by Erlanger and associates. s T h e procedure involves incubation of test serum, buffer, enzyme, and the chromogenic substrate benzoyl arginine p-nitroanilide. Results of colorimetric assays of the p-nitro-aniline released were compared with values obtained with corresponding enzyme concentrations in the absence of serum.
Alpha-1 antitrypsin and alpha-2 macroglobulin were measured by agar gel immunodiffusion on commercially available "Partigen" plates (Behringwerke).4 A m o n g the newborn infants, these determinations were carried out on umbilical cord sera or sera obtained within a few hours of life. T h e latter specimens were excluded from Table I. RESULTS
As shown in Table I, the concentrations of alpha-1 antitrypsin and alpha-2 macroglobulin and the total serum trypsin inhibitory capacity were significantly reduced in the infants with I R D S compared with lowbirth-weight infants without I R D S and full-term infants. Serum levels of enzyme inhibitors at the time of delivery a m o n g the mothers of infants with I R D S were comparable to those of low-birth-weight infants without I R D S and full-term infants. No correlation was found between the level of enzyme inhibitors (alpha-1 antitrypsin, alpha-2 macroglobulin, or total serum trypsin inhibitory capacity) and birth weight, intrauterine growth, or sex. I n 17 infants with I R D S , two or more serum samples were obtained over a period of several hours or days. Eight of these infants
590
The Journal of Pediatrics September 1972
Evans, Keller, and Mandl
].6--
E 1.4--
-
--
@=Case 1 e:Case 2 g=Case 3
@:Case 4 e~=Case5 A:Case 6
k=Case 7 r'l:Case 8
1.2--
04
0
1
2
3
4
5 6 Hours
7
8
9
10 // 32
Fig. 1A. Serial total serum trypsin inhibitor determination with respiratory distress syndrome-fatal cases.
1.6 - -
@=Case 1 @:Case2 g:Case3
-i
1.4-
N
@=Case4 e~Case 5 A'Case 6
A=Case7 El:Case 8 II--Case 9 . . . . . . . . ~ . . . . ~
A
1.0'
-r-
_z 0.8 _0.6
~ 0.4 ~
I~1 0
2
4
i [ I i I r i i i I ] i.I 6
8 16 24 26 28 30 32 38 Hours
52
72 80 96"3
I I 5 6 Days
Fig. lB. Serial total serum trypsin inhibitor determination with respiratory distress syndrome-survivors.
died (mean weight, 1,290 Gm.), and seven of these had persistently low levels, and in some still further decreases, of total serum trypsin inhibitory capacity. Only in one fatal case (Case 4) did the infant's level rise. Of the nine survivors (mean birth weight 1,422 Gin.), six had increases in serum levels of trypsin inhibitor, two had normal levels throughout the clinical course (Cases 6 and
7), and in only one of the survivors (Case 9) did the levels remain persistently low (Figs. 1A and 1B). Alpha-1 antitrypsin was measurable in 28 of 33 samples of amniotic fluid, and total trypsin inhibitory capacity was detectable in each of the 20 samples tested. In contrast, alpha-2 macroglobulin was measurable in amniotic fluid in only 1 of 29 samples. The lower limit of "detectability is
Volume 81 Number 3
Serum trypsin inhibitory capacity and RDS
about 10 mg. per 100 ml. for alpha-1 antitrypsin and about 20 rag. per 100 ml. for alpha-2 macroglobulin. DISCUSSION The data suggest that decreased levels of alpha-1 antitrypsin, alpha-2 macroglobulin, and total serum trypsin inhibitory capacity may be of diagnostic significance in IRDS. Levels of serum alpha-1 antitrypsin and total serum trypsin inhibitory capacity found in neonates with I R D S correspond to those reported in adults with heterozygous and, in some cases, homozygous alpha-1 antitrypsin deficiency. Levels found in low-birth-weight infants without IRDS, full-term infants, and mothers of neonates with I R D S correspond to values reported in normal adults. 5"7 Levels in the low-birth-weight infants without I R D S were not significantly different from those observed in the full-term infants. Alpha-2 macroglobulin levels observed in our laboratory were higher among full-term newborn infants, low-birth-weight control infants, and mothers than those recently reported by Howie and associates s in adult women. Hyvarinan and associates 9 found no difference in either serum alpha-1 antitrypsin or alpha-2 macroglobulin concentrations between low-birth-weight infants with or without IRDS. Normal serum levels of alpha-2 macroglobulin and inter-alpha inhibitor were reported among adults with alpha-1 antitrypsin deficiency? ~ Persistent low serum trypsin inhibitory capacity in newborn .infants with I R D S may be of prognostic significance. The observations reported are not completely controlled, since the patients with persistently low levels died in 10 hours or less, whereas survivors were studied for several days. Further studies are needed before definite conclusions are warranted. However, it is of interest that levels can change with time during the first few days of life and that increases appear to be associated with improved prognosis, and vice versa. The data confirm our previous findings" and those of others 9 that immaturity, per se, does not seem to predispose to serum trypsin
59 1
inhibitor deficiency. Familial predisposition to I R D S has been previously reported, al, 12 but normal levels of total serum trypsin inhibitory capacity, alpha-1 antitrypsin, and alpha-2 macroglobulin observed at term in mothers of newborn infants with I R D S are not indicative of a genetic determinant of inhibitor deficiency. Studies of phenotypes of the probands and of trypsin inhibitor levels in siblings and fathers and in mothers 6 weeks postpartum or later have not been completed. Normal values in maternal sera at term do not rule out possible heterozygous deficiency, since titers are known to be elevated at that time? s The presence of alpha-1 antitrypsin in amniotic fluid suggests that fetal synthesis, or passive acquisition, occurs early in gestation. This may allow early screening for the deficiency in mothers at high risk. The apparent absence of alpha-2-macroglobulin may be due to relatively delayed synthesis or passive transfer. The authors wish to thank Mrs. Yvonne Hosannah, Miss Mabel Wong, and Mr. Solomon O. Akpata for carrying out the serum enzyme inhibitor tests. Dr. Michael Levi performed the amniocenteses. REFERENCES
1. Klaus, M. It.: Respiratory function and pulmonary disease in the newborn, in Barnett, H., editor: Pediatrics, ed. 14, New York, 1969, Appleton-Century-Crofts, Inc., pp. 1422-1435. 2. Evans, H. E., Levi, M., and Mandl, I.: Serum enzyme inhibitor concentrations in the respiratory distress syndrome, Am. Rev. Resp. Dis. 101: 359, 1970. 3. Erlanger, B. F., Kokowsky, N., and Cohen, W.: The preparation and properties of two new chromogenic substrates of trypsin, Arch. Biochem. Biophys. 95: 271, 1961. 4. Mancinl, G., Carbonara, A. O., and Hereroans, J. F.: Immunochemical quantltation of antigens by single radial immunodiffusion, Immunochemistry 2: 235, 1965. 5. Lieberman, J.: Heterozygous and homozygous alpha-1 antitrypsin deficiency in patients with pulmonary emphysema, N. Engl. J. Med. 281: 270, 1969. 6. Maklno, S., Chosy, L., Valdivia, E., and Reed, C. E.: Emphysema with alpha-1 antitrypsin masquerading as asthma, J. Allergy 46: 40, 1970.
59 2
Evans, Keller, and Mandl
7. Kueppers, F.: Immunologic assay of alpha-1 antitrypsin in deficient subjects and their families, Humangenetik 5: 54, 1967. 8. Howie, P. W., Mallinson, A. C., Prentice, C. R. M., Home, C. H. W., and McNicol, G. P.: Effect of combined estrogen-progestogen oral contraceptives, estrogen and progestogen on antiplasmin and antithrombin activity, Lancet 2: 1329, 1970. 9. Hyvarinan, M., Graven, S. N., and Stiehm, E. R.: Development of fibrinolytic proteins in health and disease, Proc. Soc. Pediatr. Res., 1970, p. 86. 10. Ganrot, P. O., Laurell, C. B., and Eriksson,
The ]ournal of Pediatrics September 1972
S.: Obstructive lung disease and trypsin inhibitors in alpha-1 antitrypsin deficiency, Scand. J. Clin. Lab. Invest. 19: 205, 1967. 11. Graven, S., and Misenheimer, H.: Respiratory distress syndrome and the high risk mother, Am. J. Dis. Child. 109: 489, 1965. 12. Graven, S., Opitz, J. M., and Harrison, M.: The respiratory distress syndrome, Am. J. Obstet. Gynecol. 96: 909, 1966. 13. Sciarra, J. J., Chao, S., and Mandl, I.: Quantitative assay of elastase inhibitor in serum during pregnancy, Am. J. Obstet. Gynecol. 86: 753, 1963.