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sphingomyelin ratios in tracheal and pharyngeal aspirates in respiratory distress syndrome
FETAL AND NEONATAL M E D I C I N E RichardE. Behrman, Editor
Lecithin/sphingomyelin ratios in tracheal and pharyngeal aspirates in respiratory distress syndrome A preliminary report
The ratio o f lecithin to sphingomyelin (L/S ratio) in amniotic flui d has been used to determine lung maturity. However, the L/S ratio has not been determined postnatally in either tracheal or pharyngeal fluid o f itzfants with RDS. In this report L/S ratios in tracheal and pharyngeal aspirates o f 30 infants have been correlated with their clinical state. Eight it~fants with no pulmonary disease (Group 1) had ratios o)"~ I in 98% o f 45 determinations. Four it!rants with pulmonary diseases other than R D S (Group II) had ratios o f ~ 1 in 100% o f 28 determinations. Five irdant~ wilh moderate RDS (Gloup 111) had ratios o f ~ 1 in 94% o f 15 determinations. Thirteen #~fants with severe R D S (Group IV) had ratios Q f ~ 1 in only 53% o f lOl determinations; 35% o f the ratios in this group were zero. These preliminary results indicate that in[ants with severe R D S (Group IV) have a signO~eantly higher number o f pulmonary effluent L/S ratios below 1 than Ocfants in Groups 1, I1, and l l l (17< 0.01) and that infants who have ratios o f zero on two or mote occasions have a signO~cantly increased risk o f death (p ( O.05). Based on the rel;uionship o f the L/S ratio to postpartum age, survival or death was predicted correctly in 15 o f 18 infants and in ha[f o.f them t,~eprediction would have been made within 48 hr. The results o f this study suggest that L/S ratios on pulmonary effluent are useful in predicting outcome from RDS but do not appear to be o f value as a spec!)qc diagnostic test.for the disease.
Thomas A. Blumenfeld, M.D.,* John M. Driscoll, Jr., M.D., and L. Stanley James, M.D., New York, N. Y.
THE FETAL LUNG produces lecithin, a surface tension-lowering phospholipid. 1This material is present in the amniotic fluid and the L/S ratio in this fluid is used to assess fetal lung maturity and risk of developing RDS. This test is based on evidence that RDS is secondary to a deficiency of lecithin production by the fetal lung, and that near 35 wk gestation there is increased pulmonary lecithin and stable sphingomyelin production resulting in an increased L/S ratio in amniotic fluid, 2 During fetal From the Division o f Perinatology, Babies Hospital, The Children's Medical and Surgical Center at the ColumbiaPresbyterian Medical Center Supported by National Institutes o f Health Grant No. H L 14218. An abstract of this material waspresented at thejoint meeting qf the American Society of Clinical Pathology and the College of American Pathologists, Los Angeles, Calif., March 7, 1974. 9 *Reprintaddress: Department of Pediatrics, 630 W. 168th St., New York, N. Y. 10032.
life and after birth, pulmonary effluent is present in the trachea and reaches the nasopharynx. 3-5The percentage of lecithin and sphingomyelin in alveolar lung wash in infants dying from RDS and from other causes has been determined in a small series6; if the L/S ratio is calculated from percentages given, the ratio is lower in those inAbbreviations used L/S ratio: lecithin/sphingomyelin ratio RDS: respiratory distress syndrome fants dying from RDS. It has also been observed that in infants with the most severe RDS, the lecithin in their tracheal aspirates disappears; with recovery it reappears in the aspirates, s The purpose of this study was to perform L/S ratios on tracheal and pharyngeal aspirates of infants with and without pulmonary disease to determine if the ratios would be of diagnostic and/or prognostic significance.
Vol. 85, No. 3, pp. 403-407
404
Blumenfeld, Driscoll, and James
4O 50 2O I0 0
Z 0 p.
40 30 20 I0 0
hi I-hi r~
40
LL
2O
0
I I
The Journal of Pediatrics September 1974
N o P u l m o n a r y Disease
Pulmonary Disease Other Than IRDS
Mild- Moderate IRDS
30 I0 0
40
Severe ZRDS
~176 E 2O IO 0 0 >00,5 "r
I
2
:;:;ii:~:l:.~:~:!:?~ 3 4 5 6 7 8
9
__ I0 II 1 2 1 3 1 4
L/S RATIO Fig. 1. Bar graph showing the relationship of the per cent of the number of determinations from each group to the L/S ratio.
paper (Whatman IPS) and were concentrated under nitrogen in a 60~ water bath. To the evaporated residue 2 drops were a d d e d followed by 0.75 ml o f ice-cold acetone. The tubes were placed in an ice bath for 15 to 30 rain., then centrifuged. The acetone-soluble fraction was decanted and the acetone precipitate was dried with nitrogen. The dried precipitate was dissolved in 30 h of chloroform and applied to an activated silica gel plate (Quantum Industries LQD). Lecithin and sphingomyelin standards (Sigma Chemical Company) were applied adjacent to the specimen. The plates were developed in chloroform/methanol/sodium borate, 154/56/10 (v/v/ v) for a distance of 12 cm. After being developed, the plates were dried in room air for 2-3 rain and placed in a 100~ oven for 2-3 rain. After cooling, the plates were s p r a y e d with 3% c u p r i c acetate s o l u t i o n i n 8% phosphoric acid 7 and placed in a 135%C oven for 4-6 rain for charring. The presence of neutral lipids on the chromatograms was taken as evidence of adequate sampling. 5 T h e L/S ratios were c a l c u l a t e d after the chromatographs were scanned with a densitometer (Schoeffel Instrument Corporation model SD 3000) The results were stated as the closest whole n u m b e r if the ratios were greater than 0.5. RESULTS
METHODS
AND MATERIALS
P u l m o n a r y e f f l u e n t was o b t a i n e d f r o m the oropharynx or trachea of 30 newborn infants in the Babies Hospital intensive-care nursery. In most cases the samples were aspirated every 6 hr. I n f a n t s with e n d o t r a c h e a l tubes had p u l m o n a r y effluent obtained by placing into the endotracheal tube a suction catheter attached to a 5 ml syringe containing 0.5 ml of sterile saline. The saline was instilled, and the tracheal fluid i m m e d i a t e l y a s p i r a t e d by the syringe. Oropharyngeal aspirates were obtained from infants not intubated and were collected by a suction tube with attached 5 ml syringe. The first specimen was obtained within 6 hr after birth in 20% of the cases, within 12 hr after birth in 40% of the cases, within 24 hr after birth in 66% of the cases, and within 48 hr in 87% of the cases. The aspirated material was placed in a labeled, stoppered, glass tube and immediately placed in a freezer. In some instances samples were too small for analysis and were pooled with the subsequent specimen. The lipids were extracted from the aspirated material by emulsifying (vortex spinner) each specimen in 1 ml of saline, 1 ml of methanol, and 1 ml of chloroform. The tubes were placed in an ice bath until centrifugation at 3,000 rpm (1,500 g) for 5 rain. The lipids in the lower chloroform layer were separated by phase separation
Based on clinical evidence of pulmonary disease, 30 infants were placed in one of four groups (Table I and Fig. 1). In Group I, eight infants, (940-2,900 gin) gestational age 30 to 37 wk, without pulmonary disease, had a clinical diagnosis of maternal drug addiction, maternal diabetes, h y p o g l y c e m i a , sepsis, or necrotizing enterocolitis. Forty-five L/S ratios were performed and ranged from 0 to 12. One third of these specimens were collected within 48 hr after birth and collections were continued for 1 to 13 days (mean, 5). Of these ratios, 98% were I> I. Six of these infants lived and two died. In Group II, four infants (2,055-3,550 gm), gestational age 35 to 40 wk, with pulmonary disease other than RDS, had clinical diagnosis of meconium aspiration or intrauterine pneumonia. Twenty-eight L/S ratios were performed and ranged from 1 to 6. One fourth of the specimens were collected within 48 hr after birth and collections were continued for 1 to 19 days (mean, 6). All of the ratios were ~ 1. Three of these infants lived and one died. In Group III, five infants (1,758-2,330 gin), gestational age 32 to 36 wk, with mild to moderate RDS, all fulfilled the definition o f the disease both clinically and radiologically. These infants required 02 therapy or 02 therapy with continuous positive airway pressure but not mechanical ventilation. Fifteen L/S ratios were per-
Volume 85 Number 3
L / S ratios in R D S
405
Table I, Descriptive data and test results for each group of patients in the study
//.
L No pulmonary disease
Pulmonary disease other than RDS
IlL RDS (mild to moderate)
IV. RDS (severe)
Birth wt. (gm) Gestation (wk)
940-2,900 30-37
2,055-3,550 35-40
1,758-2,330 32-36
1,077-2,250 28-35
No. of patients
8
4
5
13
No. of determinations
45
28
15
101
Range of L/S ratios
0-12
1-6
0-10
0-14
0 2 0 1 0 1
1 1 1 0 1 1
5 2 4 1 0 1
15t45 (33%)
7t28 (25%)
10/15 (66%)
30/101 (30%)
1-13 (5)
1-19 (6)
1-3 (2)
1-21 (8)
1 (6%) 0 0 4 (27%) 10 (67%)
35 (35%) 8 (7%) 5 (5%) 17 (16%) 36 (37%)
5 0
5 8
Patient age at collection of first specimen (hr) 0-6 0 7-12 1 13-24 3 25-36 2 37-48 1 > 48 1 No. of specimens collected during the first 48 hr of life No. of days of specimen (mean) collection
Number and per cent of L/S ratios for each category and group 0 I (2%) 0 <0.5 0 0 0.5 0 0 1 8 (17%) 12 (43%) >1 36 (81%) 16 (57%) Survivors Deaths
6 2
formed and ranged from 0 to 10. Two thirds of the specimens were collected within 48 hr of birth and collections were contint~ed for 1 to 3 days (mean, 2). Of these ratios, 94% were >I 1. All of these infants lived. In Group IV, 13 infants (1,077-2,250 gin) gestational age 28 to 35 wk, with severe RDS, all required mechanical ventilation. A total of 101 L/S ratios were performed and ranged from 0 to 14. One third of the specimens were collected within 48 hr of birth and collections were continued for 1 to 21 days (mean, 8). Only 53% of the ratios were >1 1 and 35% were zero, i,e., contained no lecithin. Five of these infants lived and eight died. Of the infants without RDS (Groups I and II), 72 of 73 measurements of L/S ratios were one or more, 94% of the ratios ranged between 1 and 7, and three only were greater than 7. These data suggest that our normal range for L/S ratio in tracheal or pharyngeal aspirates lies between 1 and 7. Infants with clinically mild to moderate RDS had higher ratios than infants with severe RDS, the values being similar to those in infants without RDS (Group I
3 1
and II) (Fig. 1). By comparison, infants with severe RDS (Group IV) had a significant number of L/S ratios less than 1, distinguishing this group from the other three. DISCUSSION The objective of this study was to determine whether the ratio of lecithin to sphingomyelin in the pulmonary effluent after birth might be useful in the diagnosis or prognosis of RDS. Because L/S ratios in Groups I, II, and III were similar, they do not appear to be of value in the diagnosis of mild RDS or in distinguishing it from other pulmonary disease. However, the ratio is low in those infants with severe RDS and is therefore of value in identifying sick infants in a more precise manner and distinguishing them from those with mild RDS or other pulmonary disease. It should be noted that in this series infants with mild to moderate RDS tended to be more mature and their samples were obtained later in the clinical course than those with severe RDS. For these reasons it is possible that lecithin production may have been transiently decreased early in their illness before
406
Blumenfeld, Driscoll, and James
The Journal o.fPediatrics September 1974
Table II. Prediction of survival or death of infants with RDS based on relation ship of L/S ratio and postpartum age Infants with cl&ical RDS Predicted survival (ratio constantly > 1 or < I increasing to ~ 1)
Predicted death (ratio constantly < 1 or 1 decreasing to < 1)
Total cases
Prediction correct Prediction incorrect
10 3
5 0
15 3
Total cases
13
5
.18
Test resul~
Table III. Relationship of serial L/S determinations to the symptomatology of RDS and outcome Serial LIS determinations
Continuously >~ 1 Initially I> decreasing to < 1 Initially < 1 increasing to t> 1 Continuously < 1
Survival
Deaths
7 0 3 0
3* 3 0 2
*Pertinent data of infants who died: 1. 1,650gin, 30 wk gestation; severe RDS, 6 L/S ratios determined, values 3-7, last samples 20 hr before death. Autopsy: hyaline membrane disease (reparative phase), pulmonary hemorrhage, pneumonia. 1,077 gin, 28 wk gestation; severe RDS; 13 L/S ratios determined, value 1-9, last sample 1 hr before death. Clinically:sepsis, seizures, necrotizing enterocolitis, severe RDS. Twin "B," 1,300gin, 29 wk gestation; severeRDS; 1 L/S ratio, determined value 1 (3 combined specimens); last sample 4 hr before death. Autopsy: hyaline membrane disease, pneumonia, pneumothorax. (Twin A, 1,250 gin, survived,no RDS; 13 L/S ratios determined, values 1-12.) samples were obtained. It is also possible that in this group the pathophysiology is due not to a quantitative deficiency of lecithin but to lecithin transiently in a state not giving optimum decrease in surface tension. One recent study has indicated two forms of surfactant inactivation, one of which is partially reversible. 8 Current studies are in progress to obtain more information about lecithin production in mild to moderate RDS. A further possibility is that the symptomatology in infants with mild to moderate RDS may not be due to the same cause as in those infants with severe RDS. With regard to prognosis, measurement of L/S ratio proved to be of value. If two or more determinations had no detectable lecithin, the infant had a significant chance of dying (P < 0.05). Ten of 18 infants with RDS lived and only one of them had two or more determinations without detectable lecithin. In the eight infants who died, there were five who had no detectable lecithin on two or more occasions. Serial L/S determinations were also found to be useful
as a guide in predicting outcome for RDS (Table II). When the ratio was consistently one or higher, or if it was initially low, but increased to one or greater, it was indicative of survival in 10 of 13 cases. On the other hand, if the ratio was consistently less than one, or decreased to this low level, it was predictive of death in five of five cases. Thus, the outcome could have been predicted in 15 of 18 cases from serial measurements. In those patients w h e r e samples of p u l m o n a r y e f f l u e n t were taken during the first 48 hr, just over half were predictive o f outcome (nine of 16). Subsequent samples correctly predicted outcome in four of 16 patients and were not useful in the remaining three. Infants without pulmonary disease represent a population with normal lecithin production and in this group 98% of the ratios were 1 or greater and 92% of the values were between 1 and 7 (Fig. 1 and Table I). It appears that an L/S ratio of 1 or greater in our laboratory indicates adequate lecithin production in infants without lung disease and that ratios between 1 and 7 can be expected in more than 90% of determinations from this population. The L/S i'atios in infants with pulmonary disease other than RDS were very similar to values from infants without pulmonary disease (Fig. 1). In this group neither meconium aspiration nor intrauterine pneumonia had any effect on lecithin production. It is unlikely that any dilution of the pharyngeal secretions with saliva could have in any way influenced the findings in this study since the ratio was being perf o r m e d and lecithin and s p h i n g o m y e l i n would have been equally diluted. Furthermore, all of the samples in those patients with severe RDS were directly from the trachea, as were a majority of the samples in those with mild to moderate RDS. With regard to etiology, if deficiency of lecithin was the specific cause of RDS, all infants with the disease should initially have subnormal L/S'ratios (i.e., <1) in their pulmonary effluent. Those surviving should have low initial L/S ratios which increase to normal, while
Volume 85 Number 3
those dying should have ratios which are low or decrease to zero. The relationship between symptomatology of RDS and L/S ratio is presented in Table III. Of the 18 infants with RDS, 13 had initial ratios that were equal to or greater than 1; 10, including two who were sufficiently ill to require artificial ventilation, never had a ratio of less than one. Of the 10 surviving infants, only three had low initial ratios ((1) which gradually increased; ratios in the remaining seven were always in the normal range (i.e., t> 1). Of the eight infants who died, only two had consistently low ratios (( 1); three had normal initial ratios (>I 1) which fell and three with normal ratios had other causes contributing to death. These findings of normal L/S ratios in some infants with severe RDS raise several questions. Although the sphingomyelin level in amniotic fluid appears to remain rather constant, it is not known if this holds true for tracheal effluent; low ratios in this effluent could be due to elevated levels of sphingomyelin rather than low levels of lecithin. Another possibility which must be considered is that there could be additional or other factors responsible for the symptoms of respiratory distress. Finally, the differences observed in this study might be due to the variability in sample size, to dilution of samples either from pulmonary or pharyngeal secretion, or to the added saline. This explanation seems improbable because of the good correlation between low ratios and death. Despite these questions, these preliminary data sug-
L/S ratios in R D S
407
gest that the L/S ratio is of value in predicting outcome and in identifying those infants with the most severe form of RDS. It does not appear to be of value as a specific diagnostic test for this condition. REFERENCES
1. 2.
3.
4.
5.
6.
7.
8.
Adams, F. A., and Fujiwara, T.: Surfactant in fetal lamb tracheal fluid, J. PEDIATR.63: 537, 1963. Gluck, L., Kulovich, M. V., Borer, R. C., Jr., Brenner, P. H., Anderson, G. G., and Spellacy, W. N.: Diagnosis of the respiratory distress syndrome by amniocentesis, Am. J. Obstet. Gynecol. 109: 440, 1971. Adams, F. H., Desilets, D J., and Towers, B.: Control of flow of fetal lung fluid at the laryngeal outlet, Resp. Physiol. 2: 302, 1967. Setnikar, I., Agostoni, E., and Taglietti, A.: The fetal lung, a source of amniotic fluid, Proc. Soc. Exp. Biol. Med. 101: 842, 1949. Gluck, L., Kulovich, M. V., Eidelman, A. I., Cordero, L., and K hazin, A. F.: 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. Adams, F. H., Fujiwara, T., Emmanouilides, G. C., and 9Raiha, N.: Lung phospholipids of human fetuses and infants with and without hyaline membrane disease, J. PEDIATR.77: 833, 1970. Fewster, H. E., Burns, B. J., and Mead, J. E.: Quantitative densitometric thin-layer chromatography of lipids using copper acetate reagent, J. Chromatogr. 43: 120, 1969. Balis,J. U., and Shelley, S. A.: Quantitative evaluation of the surfactant system of the lung, Ann. Clin. Lab. Sci. 2: 410, 1972.
Lecithin/sphingomyelin ratios in tracheal and pharyngeal aspirates in respiratory distress syndrome A preliminary report
The ratio o f lecithin to sphingomyelin (L/S ratio) in amniotic flui d has been used to determine lung maturity. However, the L/S ratio has not been determined postnatally in either tracheal or pharyngeal fluid o f itzfants with RDS. In this report L/S ratios in tracheal and pharyngeal aspirates o f 30 infants have been correlated with their clinical state. Eight it~fants with no pulmonary disease (Group 1) had ratios o)"~ I in 98% o f 45 determinations. Four it!rants with pulmonary diseases other than R D S (Group II) had ratios o f ~ 1 in 100% o f 28 determinations. Five irdant~ wilh moderate RDS (Gloup 111) had ratios o f ~ 1 in 94% o f 15 determinations. Thirteen #~fants with severe R D S (Group IV) had ratios Q f ~ 1 in only 53% o f lOl determinations; 35% o f the ratios in this group were zero. These preliminary results indicate that in[ants with severe R D S (Group IV) have a signO~eantly higher number o f pulmonary effluent L/S ratios below 1 than Ocfants in Groups 1, I1, and l l l (17< 0.01) and that infants who have ratios o f zero on two or mote occasions have a signO~cantly increased risk o f death (p ( O.05). Based on the rel;uionship o f the L/S ratio to postpartum age, survival or death was predicted correctly in 15 o f 18 infants and in ha[f o.f them t,~eprediction would have been made within 48 hr. The results o f this study suggest that L/S ratios on pulmonary effluent are useful in predicting outcome from RDS but do not appear to be o f value as a spec!)qc diagnostic test.for the disease.
Thomas A. Blumenfeld, M.D.,* John M. Driscoll, Jr., M.D., and L. Stanley James, M.D., New York, N. Y.
THE FETAL LUNG produces lecithin, a surface tension-lowering phospholipid. 1This material is present in the amniotic fluid and the L/S ratio in this fluid is used to assess fetal lung maturity and risk of developing RDS. This test is based on evidence that RDS is secondary to a deficiency of lecithin production by the fetal lung, and that near 35 wk gestation there is increased pulmonary lecithin and stable sphingomyelin production resulting in an increased L/S ratio in amniotic fluid, 2 During fetal From the Division o f Perinatology, Babies Hospital, The Children's Medical and Surgical Center at the ColumbiaPresbyterian Medical Center Supported by National Institutes o f Health Grant No. H L 14218. An abstract of this material waspresented at thejoint meeting qf the American Society of Clinical Pathology and the College of American Pathologists, Los Angeles, Calif., March 7, 1974. 9 *Reprintaddress: Department of Pediatrics, 630 W. 168th St., New York, N. Y. 10032.
life and after birth, pulmonary effluent is present in the trachea and reaches the nasopharynx. 3-5The percentage of lecithin and sphingomyelin in alveolar lung wash in infants dying from RDS and from other causes has been determined in a small series6; if the L/S ratio is calculated from percentages given, the ratio is lower in those inAbbreviations used L/S ratio: lecithin/sphingomyelin ratio RDS: respiratory distress syndrome fants dying from RDS. It has also been observed that in infants with the most severe RDS, the lecithin in their tracheal aspirates disappears; with recovery it reappears in the aspirates, s The purpose of this study was to perform L/S ratios on tracheal and pharyngeal aspirates of infants with and without pulmonary disease to determine if the ratios would be of diagnostic and/or prognostic significance.
Vol. 85, No. 3, pp. 403-407
404
Blumenfeld, Driscoll, and James
4O 50 2O I0 0
Z 0 p.
40 30 20 I0 0
hi I-hi r~
40
LL
2O
0
I I
The Journal of Pediatrics September 1974
N o P u l m o n a r y Disease
Pulmonary Disease Other Than IRDS
Mild- Moderate IRDS
30 I0 0
40
Severe ZRDS
~176 E 2O IO 0 0 >00,5 "r
I
2
:;:;ii:~:l:.~:~:!:?~ 3 4 5 6 7 8
9
__ I0 II 1 2 1 3 1 4
L/S RATIO Fig. 1. Bar graph showing the relationship of the per cent of the number of determinations from each group to the L/S ratio.
paper (Whatman IPS) and were concentrated under nitrogen in a 60~ water bath. To the evaporated residue 2 drops were a d d e d followed by 0.75 ml o f ice-cold acetone. The tubes were placed in an ice bath for 15 to 30 rain., then centrifuged. The acetone-soluble fraction was decanted and the acetone precipitate was dried with nitrogen. The dried precipitate was dissolved in 30 h of chloroform and applied to an activated silica gel plate (Quantum Industries LQD). Lecithin and sphingomyelin standards (Sigma Chemical Company) were applied adjacent to the specimen. The plates were developed in chloroform/methanol/sodium borate, 154/56/10 (v/v/ v) for a distance of 12 cm. After being developed, the plates were dried in room air for 2-3 rain and placed in a 100~ oven for 2-3 rain. After cooling, the plates were s p r a y e d with 3% c u p r i c acetate s o l u t i o n i n 8% phosphoric acid 7 and placed in a 135%C oven for 4-6 rain for charring. The presence of neutral lipids on the chromatograms was taken as evidence of adequate sampling. 5 T h e L/S ratios were c a l c u l a t e d after the chromatographs were scanned with a densitometer (Schoeffel Instrument Corporation model SD 3000) The results were stated as the closest whole n u m b e r if the ratios were greater than 0.5. RESULTS
METHODS
AND MATERIALS
P u l m o n a r y e f f l u e n t was o b t a i n e d f r o m the oropharynx or trachea of 30 newborn infants in the Babies Hospital intensive-care nursery. In most cases the samples were aspirated every 6 hr. I n f a n t s with e n d o t r a c h e a l tubes had p u l m o n a r y effluent obtained by placing into the endotracheal tube a suction catheter attached to a 5 ml syringe containing 0.5 ml of sterile saline. The saline was instilled, and the tracheal fluid i m m e d i a t e l y a s p i r a t e d by the syringe. Oropharyngeal aspirates were obtained from infants not intubated and were collected by a suction tube with attached 5 ml syringe. The first specimen was obtained within 6 hr after birth in 20% of the cases, within 12 hr after birth in 40% of the cases, within 24 hr after birth in 66% of the cases, and within 48 hr in 87% of the cases. The aspirated material was placed in a labeled, stoppered, glass tube and immediately placed in a freezer. In some instances samples were too small for analysis and were pooled with the subsequent specimen. The lipids were extracted from the aspirated material by emulsifying (vortex spinner) each specimen in 1 ml of saline, 1 ml of methanol, and 1 ml of chloroform. The tubes were placed in an ice bath until centrifugation at 3,000 rpm (1,500 g) for 5 rain. The lipids in the lower chloroform layer were separated by phase separation
Based on clinical evidence of pulmonary disease, 30 infants were placed in one of four groups (Table I and Fig. 1). In Group I, eight infants, (940-2,900 gin) gestational age 30 to 37 wk, without pulmonary disease, had a clinical diagnosis of maternal drug addiction, maternal diabetes, h y p o g l y c e m i a , sepsis, or necrotizing enterocolitis. Forty-five L/S ratios were performed and ranged from 0 to 12. One third of these specimens were collected within 48 hr after birth and collections were continued for 1 to 13 days (mean, 5). Of these ratios, 98% were I> I. Six of these infants lived and two died. In Group II, four infants (2,055-3,550 gm), gestational age 35 to 40 wk, with pulmonary disease other than RDS, had clinical diagnosis of meconium aspiration or intrauterine pneumonia. Twenty-eight L/S ratios were performed and ranged from 1 to 6. One fourth of the specimens were collected within 48 hr after birth and collections were continued for 1 to 19 days (mean, 6). All of the ratios were ~ 1. Three of these infants lived and one died. In Group III, five infants (1,758-2,330 gin), gestational age 32 to 36 wk, with mild to moderate RDS, all fulfilled the definition o f the disease both clinically and radiologically. These infants required 02 therapy or 02 therapy with continuous positive airway pressure but not mechanical ventilation. Fifteen L/S ratios were per-
Volume 85 Number 3
L / S ratios in R D S
405
Table I, Descriptive data and test results for each group of patients in the study
//.
L No pulmonary disease
Pulmonary disease other than RDS
IlL RDS (mild to moderate)
IV. RDS (severe)
Birth wt. (gm) Gestation (wk)
940-2,900 30-37
2,055-3,550 35-40
1,758-2,330 32-36
1,077-2,250 28-35
No. of patients
8
4
5
13
No. of determinations
45
28
15
101
Range of L/S ratios
0-12
1-6
0-10
0-14
0 2 0 1 0 1
1 1 1 0 1 1
5 2 4 1 0 1
15t45 (33%)
7t28 (25%)
10/15 (66%)
30/101 (30%)
1-13 (5)
1-19 (6)
1-3 (2)
1-21 (8)
1 (6%) 0 0 4 (27%) 10 (67%)
35 (35%) 8 (7%) 5 (5%) 17 (16%) 36 (37%)
5 0
5 8
Patient age at collection of first specimen (hr) 0-6 0 7-12 1 13-24 3 25-36 2 37-48 1 > 48 1 No. of specimens collected during the first 48 hr of life No. of days of specimen (mean) collection
Number and per cent of L/S ratios for each category and group 0 I (2%) 0 <0.5 0 0 0.5 0 0 1 8 (17%) 12 (43%) >1 36 (81%) 16 (57%) Survivors Deaths
6 2
formed and ranged from 0 to 10. Two thirds of the specimens were collected within 48 hr of birth and collections were contint~ed for 1 to 3 days (mean, 2). Of these ratios, 94% were >I 1. All of these infants lived. In Group IV, 13 infants (1,077-2,250 gin) gestational age 28 to 35 wk, with severe RDS, all required mechanical ventilation. A total of 101 L/S ratios were performed and ranged from 0 to 14. One third of the specimens were collected within 48 hr of birth and collections were continued for 1 to 21 days (mean, 8). Only 53% of the ratios were >1 1 and 35% were zero, i,e., contained no lecithin. Five of these infants lived and eight died. Of the infants without RDS (Groups I and II), 72 of 73 measurements of L/S ratios were one or more, 94% of the ratios ranged between 1 and 7, and three only were greater than 7. These data suggest that our normal range for L/S ratio in tracheal or pharyngeal aspirates lies between 1 and 7. Infants with clinically mild to moderate RDS had higher ratios than infants with severe RDS, the values being similar to those in infants without RDS (Group I
3 1
and II) (Fig. 1). By comparison, infants with severe RDS (Group IV) had a significant number of L/S ratios less than 1, distinguishing this group from the other three. DISCUSSION The objective of this study was to determine whether the ratio of lecithin to sphingomyelin in the pulmonary effluent after birth might be useful in the diagnosis or prognosis of RDS. Because L/S ratios in Groups I, II, and III were similar, they do not appear to be of value in the diagnosis of mild RDS or in distinguishing it from other pulmonary disease. However, the ratio is low in those infants with severe RDS and is therefore of value in identifying sick infants in a more precise manner and distinguishing them from those with mild RDS or other pulmonary disease. It should be noted that in this series infants with mild to moderate RDS tended to be more mature and their samples were obtained later in the clinical course than those with severe RDS. For these reasons it is possible that lecithin production may have been transiently decreased early in their illness before
406
Blumenfeld, Driscoll, and James
The Journal o.fPediatrics September 1974
Table II. Prediction of survival or death of infants with RDS based on relation ship of L/S ratio and postpartum age Infants with cl&ical RDS Predicted survival (ratio constantly > 1 or < I increasing to ~ 1)
Predicted death (ratio constantly < 1 or 1 decreasing to < 1)
Total cases
Prediction correct Prediction incorrect
10 3
5 0
15 3
Total cases
13
5
.18
Test resul~
Table III. Relationship of serial L/S determinations to the symptomatology of RDS and outcome Serial LIS determinations
Continuously >~ 1 Initially I> decreasing to < 1 Initially < 1 increasing to t> 1 Continuously < 1
Survival
Deaths
7 0 3 0
3* 3 0 2
*Pertinent data of infants who died: 1. 1,650gin, 30 wk gestation; severe RDS, 6 L/S ratios determined, values 3-7, last samples 20 hr before death. Autopsy: hyaline membrane disease (reparative phase), pulmonary hemorrhage, pneumonia. 1,077 gin, 28 wk gestation; severe RDS; 13 L/S ratios determined, value 1-9, last sample 1 hr before death. Clinically:sepsis, seizures, necrotizing enterocolitis, severe RDS. Twin "B," 1,300gin, 29 wk gestation; severeRDS; 1 L/S ratio, determined value 1 (3 combined specimens); last sample 4 hr before death. Autopsy: hyaline membrane disease, pneumonia, pneumothorax. (Twin A, 1,250 gin, survived,no RDS; 13 L/S ratios determined, values 1-12.) samples were obtained. It is also possible that in this group the pathophysiology is due not to a quantitative deficiency of lecithin but to lecithin transiently in a state not giving optimum decrease in surface tension. One recent study has indicated two forms of surfactant inactivation, one of which is partially reversible. 8 Current studies are in progress to obtain more information about lecithin production in mild to moderate RDS. A further possibility is that the symptomatology in infants with mild to moderate RDS may not be due to the same cause as in those infants with severe RDS. With regard to prognosis, measurement of L/S ratio proved to be of value. If two or more determinations had no detectable lecithin, the infant had a significant chance of dying (P < 0.05). Ten of 18 infants with RDS lived and only one of them had two or more determinations without detectable lecithin. In the eight infants who died, there were five who had no detectable lecithin on two or more occasions. Serial L/S determinations were also found to be useful
as a guide in predicting outcome for RDS (Table II). When the ratio was consistently one or higher, or if it was initially low, but increased to one or greater, it was indicative of survival in 10 of 13 cases. On the other hand, if the ratio was consistently less than one, or decreased to this low level, it was predictive of death in five of five cases. Thus, the outcome could have been predicted in 15 of 18 cases from serial measurements. In those patients w h e r e samples of p u l m o n a r y e f f l u e n t were taken during the first 48 hr, just over half were predictive o f outcome (nine of 16). Subsequent samples correctly predicted outcome in four of 16 patients and were not useful in the remaining three. Infants without pulmonary disease represent a population with normal lecithin production and in this group 98% of the ratios were 1 or greater and 92% of the values were between 1 and 7 (Fig. 1 and Table I). It appears that an L/S ratio of 1 or greater in our laboratory indicates adequate lecithin production in infants without lung disease and that ratios between 1 and 7 can be expected in more than 90% of determinations from this population. The L/S i'atios in infants with pulmonary disease other than RDS were very similar to values from infants without pulmonary disease (Fig. 1). In this group neither meconium aspiration nor intrauterine pneumonia had any effect on lecithin production. It is unlikely that any dilution of the pharyngeal secretions with saliva could have in any way influenced the findings in this study since the ratio was being perf o r m e d and lecithin and s p h i n g o m y e l i n would have been equally diluted. Furthermore, all of the samples in those patients with severe RDS were directly from the trachea, as were a majority of the samples in those with mild to moderate RDS. With regard to etiology, if deficiency of lecithin was the specific cause of RDS, all infants with the disease should initially have subnormal L/S'ratios (i.e., <1) in their pulmonary effluent. Those surviving should have low initial L/S ratios which increase to normal, while
Volume 85 Number 3
those dying should have ratios which are low or decrease to zero. The relationship between symptomatology of RDS and L/S ratio is presented in Table III. Of the 18 infants with RDS, 13 had initial ratios that were equal to or greater than 1; 10, including two who were sufficiently ill to require artificial ventilation, never had a ratio of less than one. Of the 10 surviving infants, only three had low initial ratios ((1) which gradually increased; ratios in the remaining seven were always in the normal range (i.e., t> 1). Of the eight infants who died, only two had consistently low ratios (( 1); three had normal initial ratios (>I 1) which fell and three with normal ratios had other causes contributing to death. These findings of normal L/S ratios in some infants with severe RDS raise several questions. Although the sphingomyelin level in amniotic fluid appears to remain rather constant, it is not known if this holds true for tracheal effluent; low ratios in this effluent could be due to elevated levels of sphingomyelin rather than low levels of lecithin. Another possibility which must be considered is that there could be additional or other factors responsible for the symptoms of respiratory distress. Finally, the differences observed in this study might be due to the variability in sample size, to dilution of samples either from pulmonary or pharyngeal secretion, or to the added saline. This explanation seems improbable because of the good correlation between low ratios and death. Despite these questions, these preliminary data sug-
L/S ratios in R D S
407
gest that the L/S ratio is of value in predicting outcome and in identifying those infants with the most severe form of RDS. It does not appear to be of value as a specific diagnostic test for this condition. REFERENCES
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Adams, F. A., and Fujiwara, T.: Surfactant in fetal lamb tracheal fluid, J. PEDIATR.63: 537, 1963. Gluck, L., Kulovich, M. V., Borer, R. C., Jr., Brenner, P. H., Anderson, G. G., and Spellacy, W. N.: Diagnosis of the respiratory distress syndrome by amniocentesis, Am. J. Obstet. Gynecol. 109: 440, 1971. Adams, F. H., Desilets, D J., and Towers, B.: Control of flow of fetal lung fluid at the laryngeal outlet, Resp. Physiol. 2: 302, 1967. Setnikar, I., Agostoni, E., and Taglietti, A.: The fetal lung, a source of amniotic fluid, Proc. Soc. Exp. Biol. Med. 101: 842, 1949. Gluck, L., Kulovich, M. V., Eidelman, A. I., Cordero, L., and K hazin, A. F.: 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. Adams, F. H., Fujiwara, T., Emmanouilides, G. C., and 9Raiha, N.: Lung phospholipids of human fetuses and infants with and without hyaline membrane disease, J. PEDIATR.77: 833, 1970. Fewster, H. E., Burns, B. J., and Mead, J. E.: Quantitative densitometric thin-layer chromatography of lipids using copper acetate reagent, J. Chromatogr. 43: 120, 1969. Balis,J. U., and Shelley, S. A.: Quantitative evaluation of the surfactant system of the lung, Ann. Clin. Lab. Sci. 2: 410, 1972.