Pediat. Res. 11: 714-720 (1977)
respiratory distress syndrome
Absence of Phosphatidylglycerol (PG) in
Respiratory Distress Syndrome in the Newborn
Study of the Minor Surfactant Phospholipids in Newborns
MIKKO HALLMAN, BERNARD H. FELDMAN, ELSA KIRKPATRICK, AND LOUIS GLUCKMO'
Department of Pediatrics, School of Medicine, University of California, Sun Diego, La Jolla, California, USA
Phosphatidylglycerol (PG) was absent from lung effluent in 41
infants with respiratory distress syndrome of the newborn
(RDS), whereas effluent from healthy control subjects of similar
gestational age contained this phospholipid (4.9 + 2.4% of lipid-
phosphorus (P), n = 32). Control infants of 28 weeks of gesta-
tion or less with various respiratory disturbances other than RDS
also had low PG (0.2 + 0.2% of lipid-P, n = 5). In RDS
surfactant complex often could be isolated from the airways
using differential and density gradient centrifugation. The mate-
rial thus obtained had promipent phosphatidylinositol (PI) (13.6
* 2.8% of lipid-P, n = 6), but no PG. Of those 18 infants who
had such surfactant even in the early stages of RDS, 13 were 35
weeks of gestation or more, 3 were offspring of diabetic moth-
ers, and 2 had severe perinatal asphyxia. In healthy control
subjects PG sometimes appeared first within an hour of birth,
but in RDS PG did not appear until recovery from RDS.
In RDS type I1 (transient tachypnea of the newborn) PG in
lung effluent also was abnormally low (1.3 * 0.6% of lipid-P, n
= 5) and PI was correspondingly prominent (9.7 + 3.6% of
lipid-P, n = 5), indicating immaturity of surfactant similar to
Surfactant with PG and PI has superior surface-active proper-
ties compared to that containing PI, but no PG. Surfactant
without PG does not seem to stabilize the alveoli of the newborn
as well as does surfactant with PG. The failure of PG appearance
following birth therefore may precipitate RDS, especially be-
yond 35 weeks of gestation.
(7, 30). Betamethasone administered to mothers in premature
labor decreases both the incidence and mortality of RDS (28).
The analysis of surfactant in amniotic fluid by means of lecithin
to sphingomyelin (L/S) ratio has proven to be a reliable way to
predict the risk of RDS (11, 13). Therefore, the deficiency in
surfactant seems to be the primary factor precipitating RDS.
Surfactant, especially that of the neonate, has a rapid turn-
over, and is removed by lymphatics and by ciliary action, neces-
sitating continuous replenishment. By far the major fraction of
surfactant consists of lecithin, prcdominantly with saturated fatty
acids (disaturated lecithin). Its de novo biosynthesis in the lung
principally takes place via the CDP-choline incorporation path-
way (8, 17, 35). The methylation of phosphatidylethanolamine
to lecithin also may be active but of relatively minor significance
in premature babies (8, 15).
Besides lecithin the surfactant complex contains other highly
characteristic components. In the adult the second major phos-
pholipid is, uniquely PG (20,31,33), whereas, in contrast to the
earlier reports, phosphatidyl dimethylethanolamine (PDME) is
absent. In the fetal rabbit PG is absent, appearing first at birth.
Another surfactant phospholipid, PI, is present in the fetus but
then decreases concomitantly with the increase in PG (21).
The lung effluent of the newborn is a source of surfactant; its
analysis enables the monitoring of the secretory function of the
alveolar epithelial cells (15). In the present investigation pulmo-
nary surfactant from the newborn was measured. In infants with
RDS, as compared to healthy control subjects, a consistent
specific difference was found in the acidic phospholipids of lung
effluent but not always in lecithin.
MATERIALS AND METHODS
Pre- and postnatal monitoring of the acidic phospholipids, PG
and PI, in lung eflluent is useful in diagnosis and follow-up of
RDS as well as in evaluation of various therapies.
The Present material included 302 samples of lung effluent
obtained from 112 newborns at University Hospital, San Diego.
The healthy patients as well as 36% of the sick patients were
born at the same hospital; the rest came from surrounding units
within 200 miles. The infants included in this study were of
appropriate sizes for their gestations (between 10th and 90th
percentiles of the Colorado intrauterine growth charts), except
for one large for dates infant with RDS and another control of
the same age, both offspring of diabetic mothers. The diagnosis
of RDS was based on typical x-ray changes, course of disease,
and physical findings (9, 12).
The specimens of lung effluent were obtained in the same way
as described earlier (15). In addition, stomach aspirates were
collected within 1 hr after birth. In six cases stomach and retro-
pharyngeal aspirates were obtained simultaneously. These sam-
ples essentially were identical in phospholipid composition.
However, the pharyngeal specimens were smaller than those
from the stomachs and sometimes were too small to be analyzed
Idiopathic RDS of the newborn is a disease of progressive
atelectasis of the lungs with consequent hypoxia and characteris-
tic pathology (9, 12). Several lines of evidence imply that in
RDS lung surfactant is deficient, thus causing the tendency for
atelectasis of the terminal airways. Babies with RDS have de-
creased compliance of their lungs (change in volume per unit
change in pressure) (2, 25). Physiochemical and biochemical
analyses indicate that the material recovered from the airways is
deficient in surfactant (3, 15). Finally, various methods for
distending the airways by artificial ventilation improve the other-
wise grave prognosis (6, 10, 18).
Surface activity and lung stability follow a developmental time
table, first evident in late fetal life (12, 14). This development
apparently can be accelerated by drugs such as corticosteroids
ABSENCE OF PG IN RDS IN NEWBORN
for phospholipids. Lung effluent after the first neonatal hour was
obtained by retropharyngeal suctioning or, when indicated, by
direct laringoscopy. Tracheal aspirates were collected from intu-
bated patients during routine suctioning. When the sample was
small visually, two or more successive aspirates were combined.
The aspirates collected in sterile tubes either were extracted
immediately for lipids or stored at -20'. Microscopic blood was
separated as follows. The sample was suspended in isotonic
saline using a Potter-Elvehjem homogenizer and layered on 1.64
M NaBr, followed by ultracentrifugation at 27,000 rpm for 1 hr
using an SW 27 rotor. The interphase between the two layers
was collected as surfactant; erythrocytes sedimented in the pellet
and some of the serum phospholipids remained above the inter-
phase. In some infants who died the alveolar wash was obtained
by endobronchial lavage with isotonic saline (15). When sepa-
, rately indicated, the surfactant was purified further using density
gradient and differential centrifugation as described previously
Lipids were extracted as follows. The lung effluent was col-
lected and isotonic saline was added to a volume of 5 ml. The
mixture was homogenized with a few strokes from a Potter-
Elvehjem homogenizer. Five milliliters of methanol and 10 ml
chloroform were added and the resulting mixture was stirred for
10 min. Individual phospholipids were isolated with two-dimen-
sional thin layer chromatography using silica gel H (E. Merck):
NH40H (10/8/2/1 .l, v/v); second dimension: chloroform-meth-
an01 H,O (6512514, vlv). If the amount of lipid extract were
sufficient another chromatogram was developed using the fol-
lowing solvents: first dimension: chloroform-methanol-58%
NH40H-H,O (130/60/5/4, vlv); second dimension: chloroform-
methanol-acetic acid-H,O (160/50/12/4, vlv). The individual
spots were quantified by measuring the phosphorus content
PG was isolated from pooled lung effluent of the newborn and
identified using the chemical methods previously described (20).
The present chromatography method clearly separates PG from
other compounds. In addition, the presumptive PG spot in
individual runs immediately gave a blue periodate-Schiff reac-
tion (34). This feature, characteristic for an a-substituted glyc-
erol, differentiates it from other phospholipids.
Disaturated lecithins were isolated as described earlier (24).
Fatty acids esterified in a and /3 positions of lecithins first were
separated by phospholipase A, (pure venom from Naja naja,
from Miami Serpentarium, Miami, Fla.) and then analyzed by
gas liquid chromatography (16). Individual fatty acid peaks were
identified using appropriate standards, and quantified according
to the weight percentage. Before analysis the lecithin samples
were divided into two parts: one for the analysis of a and P fatty
acids and the other for total fatty acids. Measurements were
acceptable only when the'sum of the fatty acids esterified in a
and /3 positions closely resembled the fatty acids from total
The "neutral lipid" fraction was isolated on DEAE-cellulose
columns as described by Gluck et al. (14). Individual lipids were
isolated on thin layer plates of silica gel H containing 5%
ammonium sulfate, as previously described (22).
Surface activity was measured using a modified Wilhelmy
COMPARISON OF LUNG EFFLUENT BETWEEN RDS AND CONTROL
The aim of this study was to obtain specimens of lung effluent
at birth, day 1, day 2, and, if the patients were intubated, just
before extubation. We often failed to get the first sample since
more than half of the patients originated from surrounding
hospitals, although in some cases four to six specimens were
obtained during the first 2 neonatal days. In healthy control
subjects or in infants with conditions other than RDS, lung
effluent pas collected within a few hours after birth.
Figure 1 shows the content of PG in lung effluent obtained
between 1 and 48 hr after birth. One case represents the mean of
one to six measurements. PG always was absent in RDS. In
control subjects of more than 28 weeks it always was present.
In control infants of 28 weeks or less PG was practically absent
in five cases,. One of those patients did not have typical RDS;
however, he developed cardiac failure with patent ductus arter-
iosus and radiologic changes similar to RDS 1 week after birth.
PG first appeared gradually during recovery. This resembled
chronic pulmonary insufficiency observed in very premature
babies (26). Two other cases also required mechanical ventila-
tion and supplemental oxygen, but did not have x-ray changes
typical for RDS. The last two cases had apnea that responded
well to theophylline. For those who survived PG appeared at 27-
29 weeks of gestation.
The percentage of PI measured in the same samples as in
Figure 1 are shown in Figure 2. In infants with RDS of less than
30 weeks of gestation, PI was significantly lower than in control
subjects of the same gestational age. In more mature age groups
this difference was not evident; in fact, PI tended to be higher in
infants with RDS than in control infants. However, beyond 30
weeks of gestation, individual variation was considerable, and
therefore PI, in contrast to PG, seemed to be of value in
differentiating between control subjects and those with RDS
only in the group of youngest infants by gestation.
Tables 1 and 2 show the total phospholipid pattern and the
fatty acids esterified to lecithin from the same samples as in the
2 5 30
Fig. 1. The content of phosphatidylglycerol in lung effluent of new-
borns from 1-48 hr after birth. Samples during recovery from respiratory
distress syndrome (RDS) are not included. 0, controls; a, cases of
RDS. Each point represents the mean of from one to six measurements
from one individual.
Fig. 2. The content of phosphatidylinositol in lung effluent of the
newborn. Other details as described for Figure 1.
HALLMAN ET AL.
Table 1. Molar contents ofphospholipirls as percentage of total lipid-phosphorus isolatedfrom lung effluent from 1-48 hr after birth1
Group 11 (control) PC Group 111 (RDS), PC Group IV (RDS) PC <
< 0.5%; PI > 7.0%
0.2 r 0 . 2 ~
12.8 + 1.5 10.8 r 1.6
2.4 r 0.6 4.0 r 1.3
77.0 r 4.8
2.2 r 0.7 4.9 r 1.1
4.8 r 0.8
0.0 2 0.0 0.0 2 0.0
0.1 + 0.1
0.2 r 0.1
0.5 r 0.3
(control), PC > 0.5%
6.0 + 2.3'
6.7 r 2.7
1.9 + 0.6
77.0 r 7.9
1.8 r 0.3
4.9 + 0.7
0.8 r 0.3
0.2 2 0.3
0.7 r 0.8
0.5%; 5 PI 7.0%
0.0 + 0.0
3.9 + 2.0
8.0 2 1.2'
58.2 r 8.7'
16.7 2 4.0'
11.1 r 2.2'
0.1 2 0.1
0.1 r 0.2
1.9 + 1.5
Only those cases were included when the material was sufficient for thin layer chromatography with the two solvent systems (see "Methods").
RDS: respiratory distress syndrome.
' Significant (P < 0.001) difference as compared to the other three groups.
3Mean +. SD.
0.1 + 0.1
74.0 + 8.5
5.6 + 0.9
0.4 + 0.5
Table 2. Percentage composition of /3 fatty acids of Iecithitzs isolated from liit~g effluent from 1-48 hr after birr11
Group I (control), PC >
Group 11 (control), PG 5 Group 111 (RDS), PI >
Group IV (RDS), PI 5
18:3 + 20:l
The amount of phosphatidylglycerol (PG) and phosphatidylinositol (PI) as a percentage of total lipid phosphorus.
Mean r SD.
Significant (P < 0.005) differcnce as compared to the other three groups.
64.8 + 7.4 64.5 + 7.4 59.1 + 8.7 40.5 + 6.73
previous figures. Table 1 includes only those cases that were
assayed for phospholipids using two different solvent systems
(see "Methods"). Four groups were included. The first repre-
sents those cases who did not have RDS and whose PG exceeded
0.5% of phospholipid-P. The second group includes those con-
trol subjects who had 0.5% or less PG. The third represents
cases of RDS where PI exceeded 7% of phospholipid-P, and the
fourth RDS with 7% or less PI. These limits for PI content are
artificial; however, in amniotic fluid PI exceeding 7% is likely to
be associated with a mature L/S ratio (23). Therefore 7% of PI
was chosen as a limit.
The contents of sphingomyelin and phosphatidylserine were
higher in infants with RDS than in the control subjects ( P <
0.001). When PI was low in those infants with RDS, their
lecithin contents also were significantly lower than in the control
subjects (Table 1). This suggests the paucity of surfactant. The
fatty acids esterified to the a position of lecithin did not differ
significantly among the four groups (cf. Reference 15). The fatty
acids in the /3 position are shown in Table 2. They were similar in
the first three groups. However, a significantly lower percentage
of saturated fatty acids was present in the fourth group compris-
ing cases of RDS with low PI. Palmitate always was the most
prominent saturated fatty acid. Representative chromatograms
are shown in Figure 3.
We further studied whether the composition of acidic phos-
pholipids in tracheal effluent at birth could reflect an infant's
future outcome. Figure 4 shows the content of acidic phospho-
lipids as a function of age after birth. The absence of PG at birth
did not necessarily indicate that RDS was inevitable, since PG
may appear during the first neonatal hour. On the other hand, in
RDS, surfactant frequently was present at birth, as indicated by
high PI levels. However, during the subsequent hours, PG failed
to appear. The underlying control mechanisms of this induction
and its failure need further study.
An attempt was made to correlate the levels of PI at birth and
the severity of RDS. The severity of the disease possibly was
milder when surfactant was found in the airways at birth (PI
greater than 7% of phospholipid-P). However, all degrees of
severity were observed even when surfactant was present (data
Figure 5 shows the content of the acidic phospholipids, L/S
ratio, and percentages of disaturated lecithins in lung effluent
during the course of one representative infant with RDS who
had surfactant at birth. L/S ratios, percentage of disaturated
lecithins, and PI content remained high but PG gradually ap-
peared first during recovery. PG frequently remained signifi-
cantly lower than in the control subjects, even after the disap
pearance of x-ray changes typical to RDS.
The total amount of phospholipids recovered from lung ef-
fluent seemed to depend mainly on the quantity of surfactant
produced, but there was considerable individual variation. At
the height of RDS little phospholipid could be recovered during
a single suctioning (days 1-2: 0.8 r 0.4 pmol, n = 4) and during
recovery from RDS (days 4-8: 3.9 +- 2.0 ~ m o l ,
suggests that the amount of surfactant in the airways correlates
with the degree of atelectasis.
n = 11). This
STUDIES ON ISOLATED SURFACTANT COMPLEX
In 20 cases the surfactant complex was isolated using a density
gradient and differential centrifugation. When the aspirates from
ABSENCE OF PG IN RDS IN NEWBORN
infants with RDS contained less than 7 % of PI (three cases),
30% or less of material was recovered as surfactant complex.
The possible source of lung effluent in these cases include serum
and membranes from destroyed epithelial lining. However, lung
Fig. 3. The fatty acids in /3 carbons of surfactant lecithins. Above:
alveolar lavage from 2-day-old baby (birth weight 1200 g, 29 weeks of
gestation) with severe respiratory distress syndrome (RDS) who died
from intraventricular hemorrhage. Below: tracheal aspirate from 2-day-
old baby (birth weight 1020 g, 28 weeks of gestation) with no RDS.
, Phosphatidylglycerol content of purified surfactant was 4.5% of lipid-
phosphorus. Tracings of gas chromatograph are shown.
B IB I 813813
Fig. 4. The content of acidic phospholipids following birth. A: no
respiratory distress syndrome (RDS). Lefr: phosphatidyglycerol (PG)
greater than 0.8% of lipid-phosphorus at birth. Right: PG equal to or
less than 0.8% of lipid-phosphorus at birth. B: RDS. Left: phosphatidyl-
inositol (PI) greater than 7% of lipid-phosphorus at birth. Right: PI
equal to or less than 7% of lipid-phosphorus at birth. PG both right and
left less than 0.8% of lipid-phosphorus. The results are the mean 2 SD
; of two to eight cases.
effluent with high PI from babies with RDS contained 50% or
more material characteristic of surfactant in density. The follow-
ing evidence further indicated that this material was surfactant.
( I ) The fatty acid pattern of lecithin was virtually identical with
human surfactant lecithin. (2) The lecithin fraction was surface
active; i.e., it decreased the surface tension below 10 dynes/cm
on the surface balance. (3) The phospholipid composition was
similar to the control group II in Table 1 (PI: 13.6 + 2.8% of
To further study the possible differences between the surfac-
tants with and without PG "neutral lipids" were assayed in
infants with RDS (PG: 0.0 + 0.0% phospholipid-P, n = 8) and
in control subjects (PG: 6.8 + 1.8% phospholipid-P, n = 5). No
significant difference between these two groups was found; cho-
lesterol (RDS: 0.09 2 0.02 pglpg phospholipids, 47.8% of
"neutral lipids"; control: 0.09 * 0.01 pglpg phospholipid
45.8% of "neutral lipids") was the most prominent component
in each case.
The physicochemical characteristics of lung effluent were
studied on a modified Wilhelmy balance using purified surfac-
tant. In RDS infants the minimum surface tensions were signifi-
cantly higher (17.2 + 1.9 dyneslcm) than in the control subjects
(12.1 + 2.0 dynes/cm). Representative measurements are
shown in Figure 6. In RDS the surface tension of purified
surfactant failed to decrease much below 20 dyneslcm, whereas
the controls showed a sharp decrease in surface tension when the
surface was compressed.
LUNG EFFLUENT IN CONDITIONS OTHER THAN RDS
Table 3 indicates the distribution of acidic phospholipids in
respiratory disturbances of the newborn other than RDS. In
subjects with pneumonia, levels of PG and PI were similar to
those of control subjects. However, in the only case of septi-
cemia due to p-hemolytic Streptococcus type B studied, there
was low PG in four consecutive samplcs, suggesting either possi-
ble arrest in maturation or else significant destruction of surfac-
tant synthesizing cells. Also, the five cases of RDS type I1
(retained lung fluid syndrome) had low PG and high PI. After
recovery the acidic phospholipids were normal (two cases).
MALE Bw 30059 37 WEEKS
0 DlSATURdTED LECITHIN
0 PHOSPHA TIDrL GL YCEROL
Fig. 5. Changes in lung effluent of an infant with respiratory distress
syndrome (RDS). Inspiratory oxygen contents (FP,)
tory pressures (EEP); lecithin to sphingomyelin (LIS) ratios on total
lipid extracts: percentage of disaturated lecithin of total lecithin; phos-
phatidylinositol (PI) and phosphatidylglycerol (PG) (% of lipid-P) and
PGIPI ratio shown.
HALLMAN ET AL.
In six cases of postnatal and perinatal asphyxia, the acidic
phospholipids were apparently normal; in one case PC was even
abnormally high. Only one of the infants was monitored at birth,
and even then had prominent PC. The two premature babies
with severe perinatal asphyxia (cases 7 and 8 with 1 min Apgar
scores of 1 and 3, respectively) did not develop PC after birth
and RDS ensued.
RDS- ABSENT PHOSPHATIDYLGLYCEROL
The virtual absence of P C from lung effluent in the newborn
with RDS was demonstrated in this study. In striking contrast
Table 3. Contet~t of acidic phospholipi~ls in licrtg efflu~nt of
neonates witlt various respiratory dkturbancesl
this phospholipid was always present in full term babies: healthy
newborns, newborns with pneumonia, and asphyxiated new-
borns. The earlier investigations of Gluck and associates (15)
suggested that a precursor of lecithin, PDME, was absent in
RDS, indicating possible absence of inhibition of synthesis of
lecithin by thz methylation pathway. It was suggested that when
the methylation pathway was absent, the choline incorporation
pathway did not provide sufficient surfactant in the premature
baby and RDS ensued.
The identification of PDME in dog lung was first reported by
Morgan et al. (29) and presumptive verification made by Cluck
et al. (16). However, recent evidence (22, 31,33) indicates that
substantial amounts of P C and not PDME are present in adult
and newborn surfactant. On the other hand, present data on the
distribution of PC essentially confirm the earlier primary find-
ings of Cluck and associates, although the interpretation of the
results is different.
% of phospholipid-P
Diagnosis PG PI
1 : Cytomegalovirus
2: Candida albicans
3: Streptococcus group B
4: Meconium aspiration
5: Meconium aspiration
RDS type I1
1 : Congenital heart disease
2: Congenital heart disease
3: Congenital heart disease
7: Perinatal + RDS
8: Perinatal + RDS
Numbers in parentheses indicate the content of acidic phospholipid
in control subjects of the same gestational age. RDS: respiratory distress
syndrome; PG: phosphatidylglycerol; PI: phosphatidylinositol.
RDS - SURFACTANT NOT NECESSARILY ABSENT
The present findings agree with earlier findings on the defi-
ciency of surfactant in the airways of most newborns with RDS
(3, 15). In these cases surfactant could be found in the airways
only after one or several days of life, its appearance heralding
the recovery phase. However, in about 40% of the present
patients sampled, surfactant complex was found in tracheal ef-
fluent even in the early stages of the disease. Of these, 72%
were of gestational age of 35 weeks or more, three were infants
of diabetic mothers, and two were born after severe perinatal
asphyxia. The clinical hallmarks and the x-ray findings essen-
tially were indistinguishable from RDS where surfactant could
not be found. The findings of surfactant in RDS agree with that
of Lauwcryns (27) and Balis et al. (4), who sometimes detected
impressive numbers of lamellar bodies apparently representing.
intracellular collections of surfactant in patients with RDS.
The present study focuses on the qualitative aspects of surfac-
tant; this could be equally as important as the quantity of surfac-
tant in stabilizing the alveoli. In normal pregnancy PI that
presumably is derived from the lung (22) increases in amniotic
fluid after the 30th gestational week. PC first appears after the
35th week (23). Birth may affect the normally gradual develop
ment of the acidic phospholipids in the fetus: in some healthy
prematures PC appeared rapidly in lung effluent within the first
neonatal hour. However, in RDS it gradually developed only
during recovery. The failure of induction of P C with prominent
PI at birth is remarkable and suggests that some regulatory
factor(s) may be defective.
20 40 GO .
PERCENT SURFACE AREA
20 40 60 80 100
Fig. 6. The recordings of surface tension-area relationship on a modified Wilhelmy balance of purified surfactant from a 37-week-old baby at
birth (lefr) and from an infant of a diabetic mother with RDS (birth weight 3500 g), 36 weeks of gestation (right). The compression of the surface is
indicated by the arrows. The amount of phospholipid applied to the surface was 30 nmol, representing 10 A~lmo~ecu~e
of the surface.
during maximal compression
ABSENCE OF PG IN RDS IN NEWBORN
In RDS type I1 (retained lung fluid syndrome) the contents of
P C were low and PI prominent. During recovery the acidic
phospholipids normalized. This suggests that the retained lung
fluid syndrome may be a mild form of RDS with somewhat
immature surfactant but sufficient in function to prevent atelec-
POSSIBLE SIGNIFICANCE OF ACIDIC SURFACTANT PHOSPHOLIPIDS
The present evidence thus far suggests that P C improves the
quality of surfactant that may be critical in stabilizing the alveoli.
The tubular airways of the bird lung (22) as well as the alveolar
ducts of the very premature lung contain little, if any, P C but
prominent PI, suggesting that the requirements for surfactant
may vary on the basis of the structure of the terminal airways.
Moreover, according to the LaPlace formula, the pressure (P)
tending to collapse the curved surface is dependent on the shape
where p = surface tension; R and r = the principal radii of the
curvature. Thus, in a sphere (alveolus), the collapsing pressure
(Pa = 2p/r) is twice as great as in a cylinder (tubular airway) of
the same surface tension and radius (P, = p/r). Further studies
are needed to establish dependence between the structure of the
terminal airways and the composition and physicochemical char-
acteristics of surfactant.
The present findings agree with the hypothesis that a defect in
surfactant is a primary factor in the pathogenesis of RDS (11,
13, 15). The amount of surfactant in the airways at the height of
the disease is always diminished. However, in some cases, such
as most of those beyond 35 weeks of gestation, the qualitative
rather than the quantitative aspects of surfactant may be the
major cause of the disease.
The rate of surfactant production and its release into the
airways increase during perinatal development (1 1, 13, 32). At
the same time the individual components characteristically
change in concentration (21, 22). The complexities of surfactant
ontogeny imply multiple control mechanisms. Studies in this
area may further refine the diagnosis as well as open new ways of
prevention and therapy.
In RDS phosphatidylglycerol (PC) was always absent in lung
effluent, whereas lung effluent from healthy newborns and those
with pneumonia or cardiovascular disease contained this phos-
pholipid. However, in some cases of RDS, even in the early
stages of the disease, surface-active lecithin was present in the
airways. The measurements on a surface balance suggest that
surfactant with PG as compared to that without PG has better
physicochemical characteristics to guarantee alveolar stability.
P C as a component of the surfactant complex serves as a
biochemical marker: in its absence there is a significant risk for
developmental lung disease; the presence of P C suggests bio-
chemical maturity of the surfactant.
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(Submitted for publication.)
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720 Download full-text
CRESTEIL AND LEROUX
ment of the rat lung. J. Lipid Res.. 9: 262 (1968).
36. Informed consent was obtained for all specimens obtained from patients.
37. The present address of Dr. Mikko Hallman is: Children's Hospital, University
of Helsinki, Stenbackinkatu 11. 00290 Helsinki 29, Finland.
38. The present address of Dr. Bernard H. Feldman is: Medical Director, South-
western Neonatal Intensive Care Unit, Sunrise Hospital, Las Vegas, Nev.
39. This research was supported by National Institutes of Health Grant HD
04380, SCOR HL 14169, TW-02051.
40. Requests for reprints should be addressed to: Louis Gluck, M. D., Depart-
ment of PediatricsIC-019, University of California, San Diego, La Jolla,
Calif. 92093 (USA).
41. Received for publication April 30, 1976.
42. Accepted for publication October 8, 1976.
Copyright 0 1977 International Pediatric Research Foundation. Inc.
Pediat. Res. 11: 720-723 (1977)
Early Postnatal Metabolism of Amino Acids in
THIERRY CRESTEIL'2" AND JEAN-PAUL LEROUX
Laboratoire de Biocl~imie, INSERM U-75, CHU Necker-Enfants Malades, Paris, France
The production of 14C0, from glutamate and alanine studied
in the newborn rat after injection of labeled precursors was
intense and revealed an important catabolism from 130 nmollhr
in a 0-hr neonate to 2000 nmollhr in 3-week-old rat. The
degradative metabolism of leucine was low at birth (10 nmollhr),
but increased up to 500 nmol/hr in 3-week-old rat. Phenylala-
nine was practically not metabolized into CO,, but its incorpora-
tion into protein was higher than leucine, alanine, and glutamate
incorporation. Glucose is the major fuel utilized by newborn rat
for it8 energy supply (300-560 "mollhr). ow ever, the relative
part of amino acids in energy production is important in the early
neonate (0 and 1 hr after birth).
In the early newborn rat, the important oxidative metabolism
of amino acids (before phosphoenolpyruvate carboxykinase
(PEPCK) biosynthesis and gluconeogenesis activation) indicates
that they may be directly utilized for energy requirements. This
amino acid oxidation could be considered as a transient energetic
pathway in species showing a delayed development of PEPCK
activity (e.g., the human neonate).
In the fetus, the energy supply is provided by the mother
through the placental barrier. Amino acids and glucose are the
two major nutrients which cross the rat placenta: freely for
glucose (18) or by an active transport mechanism for amino acids
(2). Maternal glucose appears to be the major oxidative fuel for
the fetus in utero (I), but it is also utilized during the last days of
gestation for the constitution of endogenous stores: hepatic gly-
cogen and triglycerides in brown adipose tissue. Amino acids are
essentially used for macromolecule synthesis.
However, metabolic measurements in fetal lamb indicate that
amino acids are also utilized for energy supply. The fetal urea
production (a reflection of amino acid catabolism) and its excre-
tion to the maternal circulation support this view (9).
Printed in U.S.A.
In the first hours following birth, as during starvation, a
transient hypoglycemia is observed; carbohydrate stores are then
utilized for energy requirements whereas endogenous triglycer-
ides assure thermogenesis in brown adipose tissue. An active
gluconeogenesis from amino acids appears after some hours and
maintains constant the plasma glucose concentration.
In the present study, the respective participations of amino
acids in oxidative processes (directly or after conversion into
carbohydrates) and in protein biosynthesis were investigated in
vivo during the early postnatal period.
MATERIALS AND METHODS
Newborn rats (Sprague-Dawley strain) were obtained by ces-
arian section on the 21.5th day of gestation under light ether
anesthesia. Metabolic measurements have been performed pre-
viously on control animals delivered without anesthesia; no sig-
nificant differences in the 14C02 production were noted and the
values were pooled. Labeled precursor (0.5 or 1 pCi) was
injected intraperitoneally and the respiratory 14C0, was moni-
tored for 1 hr starting immediately after injection. The relative
concentrations of precursors were related to the plasma concen-
trations given for newborn rat in the literature. Tracer amounts
of precursors were injected in a 50-p1 volume for newborn
animals or 500 pl for 3-week-old rat: L-[U-14C]alanine, 3 mM;
L-[U-14C]glutamate, 2 mM; L-[U-14C]leucine, 1 mM; L-[U-
'4C]phenylalanine, 0.5 mM; D-[U-14C]glucose, 40 mM.
After injection, animals were placed in a sealed dessicator (or
flask) kept at 37' into which humidified air was flowed at con-
stant pressure. CO, was collected in NaOH (10 N) and con-
verted into BaCO, for radioassay with Unisolve as scintillator.
At the end of incubation, blood was collected, tissues were
quickly excised, weighed, and homogenized in 5% cold trichlo-
roacetic acid. After centrifugation proteins were dissolved in 3
ml 88% formic acid to which 0.6 ml 30% hydrogen peroxide
was added. The reaction was carried on 30 min under magnetic
stirring (13). Proteins were reprecipitated with trichloroacetic
acid, added to Unisolve, and counted in a Packard scintillation
spectrometer with quenching standardization.