Abnormal
Lung
Surfactant
Related
to Essential
Fatty
Acid Deficiency
in a Neonate
Zvi Friedman, M.D., F.R.C.P.(C), and Abraham Rosenberg, Ph.D.
From,, tlt’ 1)Eiartmllents of Pediatrics and Biological Clienistry, TI,e Milton S. Ilershey ‘stedieal Ce,,ter of tle
Penn.sylrania St(It(’ t mirersity College of %fedicine, Hershey, Pen n.sylrania
ABSTRACT. A low-birth-weight infant, suffering from
chronic bronchopulmonar dysplasia following hyaline
mcI1l)rane disease and recurrent episodes of necrotizing
enterocolitis, developed biochemical evidence of essential
fatty acid (EFA) deficiency in the plasma. Fatty acid
(01111)OsitiOfl of phosphatidlcholine and
phosphatidlglcer-ol in the lung lavage fluid was abnornial. Plasma changes
includcd a decrease in the level of linoleic acid and an
increased level of palmitic, palniitoleic, oleic, and
5,8,11-eicosatrienoic acids, the ratio of 5,8,1 1-eicosatrienoic acid to
arachidonic acid being >0.4:1. A lower than normal level of
palmitic acid and an increased level of palmitoleic and oleic
acids were SCCII in )ulmonar surfactant phospholipid
C0IU1)OfleIlts. U1)()n treatment and recovers’ from EFA
deli-ciencv, the fatty acid pattern both in plasma and surfactant
l)1i5l)110l ipids returned to normal along with clinical
improvement. Au association l)etween EFA deficiency and
altered fatty acid composition of pulmonary surfactant
phospholipids is suggested. Pediatrics 63:855-859, 1979,
(‘s.s(’nti(Il fatty (1(i(l (lefu-iency. lung surfactant.
The presence of a surface tension-lowering material in lung alveoli was first suggested by von Neergaard in 1929.’ In 1946, Thannhauser et al2
observed the high content of saturated lecithin in
lung tissues. Soon others’ began to realize that
the lung is an active site of lipid synthesis and that, in the alveolar lung fluid, an insoluble film
forms at the air-liquid interface and modifies its surface tension. Clements et al later
demon-strated that this interfacial material reduces the
pressure across the alveolar interface, thereby
promoting alveolar stability. King and Clements7
also have shown that 85% of this surfactant
material is lipid, of which approximately 75% by
weight is phosphatidlcholine. The major molec-ular species of phosphatidylcholine in the lung
and lung lavage fluid is dipalmitoyl glycerophos-phocholine, which plays an important role in determining the surface properties of pulmonary surfactant .
Essential fatty acid deficiency is reported to
cause respiratory disease syndrome in chickens”;
in rats, it results in a significant reduction in the patmitate content of lung surfactant. These changes have been associated with impaired surface tension-lowering activity of this materi-al.i)2 Development of EFA deficiency has been described in infants and children who have been maintained on fat-free diets’ and fat-free intraye-nous alimentation.’
The present studies were undertaken to ascer-tam in a human subject, an EFA-deficient infant with chronic bronchopulmonary dysplasia
follow-ing hyalmne membrane disease, whether there was
a reduction in palmitate content of the lung lavage phospholipids and to investigate the time
course of reversal of this alteration once the infant is fed a diet containing linoleic acid. The results
show a decrease in palmitate content of lung lavage phospholipids during the EFA-deficiency
state in a human infant. On feeding the infant a
lmnoleate-containing diet, reversal was completed within one month.
MATERIALS AND METHODS
Case Report
The infant was born to a 22-year-old gravida 3, para 2
black woman after spontaneous labor associated with
al)rup-tio placentae at 30 weeks’ gestation. Apgar scores were 2 at
one and ten n#{236}inutes,respectively. The infant was
resusci-tated vigorously and later transferred to the Neonatal
Intensive Care Unit at MSHMC. Birth weight was 1,300 gin
(40th percentile); length, 41 cn (60th percentile); and head
circumference, 27.5 cm (40th percentile). The infant
devel-oped respiratory distress that required endotracheal
intuba-Received July 20; revision accepted for publication October
19, 1978.
ADDRESS FOR REPRINTS: (Z.F.) Department of
Pediat-ncs, Baylor College of Medicine, 1200 Motirsund Avenue,
3000
25001-:
2000 I1500
1000L
150
125
100
75
50
25
0
Parenteral
I
Nutrition
Portagen
I I I I I I . . ‘
1
1 2 3 4 5 6 7 8 9 10 11 12 13 14
WEEKS OF LIFE
Nutritional regimen and weight gain in patient during postnatal life.
C’,
---S-0 -o1:)
tiol3 and mechanical ventilation. Hpotension and anemia
were corrected with blood transfusions. The clinical course
was complicated l)\ patent ductus arteriosus, convulsions,
chronic bronchopulmonar dsplasia, and cor pulmonale.
Recurrent episodes of necrotizing enterocolitis required the
administration of parenteral nutrition. The intravenous
solu-tions included dextrose, electrolytes, a parenteral
alimenta-tion mixture containing a synthetic solution of aniino acid,
trace minerals, and vitamins. Fluid intake varied between 75
and 220 ml/kg/24 hr and caloric intake between 18 and 135
kcal/kg/24 hr. Oral feeding included Portagen, a formula
containing lipid mainly in the form of medium-chain
trigly-cerides (MCT) and a sniall amount of corn oil containing
linoleic acid.
The feeding regimen is illustrated in the Figure. The
infant died at 3#{189}nonths of age. Permission for postmortem
examination was refused.
Lipid Analysis of Plasma and RBC
Whole blood, anticoagutated with EDTA, was obtained via a central line or peripheral veni-puncture and centrifuged at 4 C. The plasma and
RBCs were frozen and stored in 100% nitrogen
until lipid extraction was begun. The various lipid fractions-phospholipids, cholesterol esters, tn-glycerides, and free fatty acids-were separated
by thin-layer chromatography, and the fatty acid composition of each lipid fraction was then
deter-mined by gas-liquid chromatography. These methods have been described previously.’
Lung Lavage
Following chest physiotherapy, lung lavage was
obtained using a Fr 6 suction catheter via an
endotracheal tube. Three washes, each utilizing 0.5 ml of normal saline, were collected. The material was centrifuged within 30 minutes at 101 x g for 15 minutes and pellet (A) and float (B) were removed. The remaining supernatant was centrifuged at 10
x
g for 60 minutes and pellet(
C) was separated from supernatant (D). Thesefractions were extracted with choloroform and methanol. ‘ The phospholipids were separated
from neutral lipids by thin-layer chromatography in a variation of a published procedure,” using chloroform, methanol, and water (65:35:8). Spots
were identified by comparison with reference
compounds exposed to iodine vapors. The fatty acid composition of the various phosphotipids was
analyzed by gas-liquid chromatography.”
RESULTS
Plasma and RBC Lipids
The infant was found to have EFA deficiency based on conventional criteria.’7 In addition to a
decrease in the linoleic acid level, there was a charactenisitic increase in 5,8,11-eicosatnienoic
acid (Table I). The ratio of 5,8,11-eicosatnienoic acid to arachidonic acid, the “trienoic/tetraenoic ratio,” is ordinarily less than 0.4:1.’ In this infant, the ratio in plasma was 0.6: 1 during EFA
defi-ciency and less than 0. 1 : 1 during recovery. The
changes in the fatty acids in the RBC phospholip-ids are less obvious than those observed in the
Lung Lavage
TABLE I
The percentage changes in the composition of the fatty acid methyl esters derived from
phos-phatidylcholine (PC) and phosphatidytglycerol (PG) fractions of the lung lavage during EFA deficiency and upon recovery are shown in Table II. During the period of EFA deficiency, there is a
decrease in palmitate and linoleate in the PC and a decrease in palmitate in the PG fraction.
Smniultaneously, there is an increase in the levels
of palmitoleate and oleate in the PC fraction and
an increase in the level of palniitoleate in the PG fraction. Upon the infant’s recovery from the EFA-deficiency state, an increase in the levels of
palniitate and linoleate and a decrease in the level of palmitoleate are seen in the PC and PG
fractions, and a decrease in the level of oleate is
seen in the PG fraction.
DISCUSSION
The patient developed biochemical changes in
the plasma that were compatible with the diagno-sis of EFA deficiency and which were reversible
with oral feedings containing EFAs. Both
arachid-onate and linoleate are effective in the treatment of EFA deficiency’’ but, as linoleic acid is readily
converted to arachidonic acid in vivo,2’ it is the
primary nutrient necesary for the prevention of EFA deficiency. There are two sources of linoleic acid: dietary fat, the prime source, and adipose tissue stores. In the fetus, the proportion of linoleic acid in tissue phospholipids increases with advancing gestational age2’ so that the prematurely born infant is endowed with limited
stores. Because of their limited nonprotein caloric
reserve, these infants must mobilize fatty acids for
caloric needs when faced with deficient dietary intake. Adipose tissue triglycerides undergo constant hydrolysis with the release of EFA and
other fatty acids to the plasma. During
hyperali-mentation, however, the outflow of linoleic acid
froni adipose tissue is blocked, at least in part by
the high insulin levels accompanying glucose
administration. During his first 7 /2 weeks of life, at which time the EFA deficiency was
detected, the patient had gained only 100 gill
over his birth weight. During this time he was
receiving fat-free parenteral nutrition that was
supplemented for a period of two weeks with
Portagen. Although the EFA composition was
deternlined for plasma lipids, tissue levels of
EFAs were likely to be reduced as well.
EFA deficiency has been shown to cause
respi-ratorv disease syndrome in chickens,” and in rats
it resulted in a significant reduction in palmitate
FATTY ACID CONTENT IN PLASMA ANI) RBC PH05PH0LIPID
FRACTION AT 7.5, 11, AND 13 WEEKS OF LIFE#{176}
Fatty Acidst % i
7.5 n Plas 11 ma 13 % 7.5 in RB 11 C 1.3
Palmitic (c16:0) 27.6 23.8 23.2 22.9 21.2 20.8
Palmitoleic (c16:1) 2.7 1.7 1.8 2.1 2.2 2.4
Stearic (c18:0) 12.2 12.6 12.9 9.6 10.8 11.4
Oleic (c18:1 (.o9) 21.0 17.2 16.8 18.7 16.8 15.9
Linoleic (c18:2w6) 6.3 19.5 18.8 8.6 12.2 12.6
5,8,11-Eicosatrienoic 6.0 1.7 0.9 1.0 0.6 0.5
(c20:w 9)
Dihomo-’y-linolenic 4.4 4.4 4.6 2.3 2.8 3.1
(c20:3 Cd6)
Arachidonic (c20:4w6) 10.0 10.8 11.6 14.9 15.2 15.6
‘EFA deficiency was greatest at 7.5 weeks of life, with
‘ecovery taking place between 1 1 and 13 weeks of life.
1The abbreviated formula indicates the number of carbon
atoms and the number of double bonds. The position of the
double bond nearest to the methyl terminus is indicated by
the symbol w.
content of lung surfactant, but not in the total
amount of phospholipids in lung tissue and lavage fluid.”
Our data show that simultaneously with EFA
deficiency in plasma, there is evidence of EFA
deficiency in pulmonary surfactant PC and PG. However, in lung surfactant, EFA deficiency is
associated with a reduction of palmitate content, whereas in the plasma the reverse is true. Once
linoleate-diets containing are fed, the time course for correcting the dietary state in the plasma and
pulmonary lavage PC and PG is similar.
Clinical-ly, upon recovery from the EFA-deficiency state, an improvement in the respiratory illness was
noted in our patient. The infant was extubated for
several days, but superimposed pneumonia and
atelectasis required reintubation and mechanical ventilation. The major molecular species of PC in lung tissue and lavage fluid is dipalmitoyl PC.
This disaturated species of PC is considered to play an important role in determining the surface
properties of pulmonary surfactant.M Reduction
in this species of PC results in surfactant with diminished surface tension-lowering capacity in
EFA-deficient rats. ‘
Beside PC, the surfactant complex contains
other highly characteristic phospholipid
compo-nents. In the adult, the second major phospholipid
is phosphatidylglycerol.2 Ill the fetal rabbit, PC is
absent, appearing first at Y’ Recently, PG deficiency has been demonstrated in infants with
TABLE II
F:rry Acm CoMPosITIoN OF LUN(; LAVA(;E PHOSPHOLIPIDS DURING
EFA DEFIcIENc- AND RECOVERY0
l1(i(ti()lif (111(1 iiIll(’
of .?l(1liJSiS
I(Itt iJ Acids
(%)1:
--
---Palmnitic Palmitoleic Str’aric Olcie Linolcie
(e16:0) (c16:1) (c18:0) (c18: 1 cv 9) (e18:2 w 6)
P!o.spliatidijleholiie
Fraction A
At 7.5 svk of life 53.6 10.3 10.4 17.8 0.4
At 11 wk of life 65.2 6.6 9.9 12.2 1.5
At 13 wk of life 68.4 6.3 9.7 14.5 1.7
Fraction B
At 7.5 wk of life 22.2 16.6 24.3 28.6 1.8
At 11 wk of life 30.9 5.9 23.0 25.1 5.1
At 13 wk of life 62.7 6.0 13.2 9.5 5.6
Fraction C
At 7.5 wk, of life 46.8 12.5 8.9 19.8 ND
1:3 wk of life 62.7 7.3 10.2 9.5 1.2
Fraction D
At 7.5 wk of life 24.1 8.9 11.8 19.5 2.0
At 13 wk of life 56.2 6.8 13.2 13.1 4.2
P!ao.sp!iatidijlglqeerol
Fraction A
At 7.5 wk of life 19.1 8.5 15.0 36.7 3.0
11 wk of life 24.2 6.3 20.0 37.2 3.0
13 wk of life 31.9 4.3 18.6 37.4 3.4
Fraction B
7.5 wk of life 22.6 18.6 19.2 33.0 2.3
At 11 wk of life :36.1 5.4 24.5 24.6 4.4
At 13 wk of life 38.3 7.8 30.1 27.8 4.8
#{176}Materials from tracheal lavage were analyzed at 7.5 weeks of life, which is the period of EFA
deficiency, and at 1 1 and 13 weeks of life, which is the period of recovery.
tAnalysis of tracheal lavage material was performed on the following fractions obtained by
centrifugation: A-pellet, 10
x
g; B--float, 10 X g; C-pellet, 10x
g; D-supernatant,10
x
g.tThe abbreviated formula indicates the number of carbon atoms and the number of double
l)OI1(ls. The position of the double l)ofld nearest to the methyl terminus is indicated by the sym-1)01 CL).
§Nl) = level hot detectable.
fatty acid composition of surfactant PG during the EFA-deficient state may further impair its surface-active tension properties.
Lung structure is not altered by EFA
deficien-cy except for mitochondnial changes confined to type II cells.’ Therefore, the alteration in lung
mechanics in EFA-deficient rats results from impaired surfactant surface tension-lowering activity.’2 Impaired surfactant activity in humans
with EFA deficiency, although likely to occur, has not yet been demonstrated.
The association between EFA deficiency and the impairment of surfactant PC and PG fatty acid composition has been shown in the present study. This alteration niay diminish lung function
and so contribute to the pathophysiolog of hyalmne membrane ‘ chronic
bronchopul-monary dysplasia, cystic fibrosis,272M and other respiratory diseases associated with inadequate
nutrition inviting an EFA deficiency. Prevention of EFA deficiency in such patients may be essential for the maintenance of normal lung function.
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ACKNOWLEDGMENT
This investigation was supported by grants No. 1 ROl HD
11255 from the National Institute of Child Health and
Human Development and No. NS08258 from the National