Biochemical Development of Surface Activity in Mammalian Lung

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Pediat. Res. 4: 352-364 (1970) , Developmental lipids biochemistry fatty acids lung lecithin surface activity Biochemical Development of Surface Activity in Mammalian Lung

III. Structural Changes in Lung Lecithin during Development of the Rabbit Fetus and Newborn

Louis GLUCIC^22!, ROBERT A.LANDOWNE and MARIE V. KULOVICH Department of Pediatrics, University of California, San Diego School of Medicine, La Jolla, California, and American Cyanamid Research Laboratories, Stamford, Connecticut, USA

Extract

This is a report of studies with gas-liquid chromatography of the fatty acids on the a- and /9-carbons of surface active and nonsurface active lecithins isolated from alveolar wash and from residual lung after wash in the developing rabbit fetus. The total fatty acids of lecithin show no clear tendency toward greater saturation with development. In alveolar wash, there was greater saturation of fatty acids (65 %) compared with total lung (57 %). Fatty acids separately determined on a- and /J-carbons of lecithins showed significant developmental differences. Differences in surface active acetone- precipitated lecithin and nonsurface active acetone-precipitated and acetone-soluble lecithin were in the ^-carbon fatty acids. They were highly saturated (+70%) in surface active lecithin but only about 25% saturated in nonsurface active lecithin. Concentration of acetone-precipitable lecithin in whole lung rises during fetal development to a peak (84%) with breathing. There was a marked increase in acetone-precipitable lecithin in alveolar wash after 1 h of breathing (0.35 mg/g dry weight lung in nonbreathing full term to 9.8 mg/g), a 30-fold increase. During gestation, both a- and ^-component palmitic acid (Ci6:0) rose abruptly after day 29 (a-45 to 67% and /S-48 to 60%) when alveolar lecithin becomes surface active. The greatest increase in a- and /S-palmitic acid followed the onset of breathing, and by day 2 of age, a-palmitic 85%, ^-palmitic 62.5%. Thus, dipalmitoyl lecithin is the greatest single identifiable fraction of surface active lecithin isolated from rabbit alveolar wash. Acetone-soluble lecithins, even in the breathing animal, have only about 25 % palmitic acid on the /3-carbon. Early 29-day fetus, delivered by cesarean section, synthesized de novo 100% of his surface active lecithin (CDP-choline +a -/S-diglyceride pathway), predominantly a- and ^-palmitic acid (69.7and 61.2%, respectively). Phosphatidyl dimethylethanolamine (PDME), a surface active intermediate in the trimethylation of phosphatidyl ethanolamine (PE) to form lecithin, showed largely an a-palmitic//?-myristic acid (53/60 %) distribution. Since the rate-limiting reaction is the first methyl group in PE, PDME represents the lecithin end product, another pulmonary surface active lecithin with a-palmitic/|8-myristic acids.

Speculation

Use of the ^-carbon fatty acids as markers in studying the acetone-precipitated surface active fraction of lecithin isolated from alveolar wash will permit an assessment of the contribution of each of the two major pathways in the biosynthesis of surface active lecithin. GLUCK, LANDOWNE and KULOVICH 353

Introduction acid composition associated with surface active lecithin synthesized by each of the two pathways are presented.

The stability of mammalian lung is greatly dependent upon a phospholipid-rich complex, which apparently Materials and Methods lines the alveoli and which lowers surface tension on expiration, thus preventing atelectasis. Quantitatively, Pooled fetal lungs from a pure strain of albino New the largest component of this lining, measured either Zealand white rabbits were studied on successive days after tracheal lavage with saline or by saline extraction of gestation, from 23 days to term (31 days). Five to of minced normal adult mammalian lung, is lecithin. eight litters for each day of gestation were examined Previous studies [8, 10, 12] have reported that sur- averaging nine fetuses per litter. All determinations face active lecithin when present in a total lecithin were done in duplicate and some were done in qua- fraction can be separated by precipitation in cold ace- druplicate. tone. The resultant acetone-soluble fraction is not Lecithin was isolated, purified, and separated into surface active when measured on a modified Wilhelmy acetone-soluble and acetone-precipitated fractions as balance although the surface active acetone-precipi- previously described [6, 8, 10]. Separation into these table fraction reduces the tension on compression of fractions involves adding chloroform in a proportion the surface film to less than 15 dynes/cm, increases the of about 1:100 (v/w) to purified lecithin to barely surface tension on expansion of the surface film, and moisten (liquify) it and then adding, drop by drop, describes a hysteresis during the cycle. In a previous an excess of cold (0°) acetone until no further precipi- report [7], comparison of fatty acid ester compositions tation occurs (in proportion of 1 ml acetone/mg leci- of acetone-precipitated surface active lecithin and non- thin) . The mixture is then centrifuged in a refrigerated surface active acetone-soluble lecithin was not made. centrifuge and the supernatant is decanted. Addition In 1961, KLAUS et al. [13] noted a similarity in sur- of more cold acetone to the precipitate and combining face activity between synthetic dipalmitoyl lecithin the precipitates is followed by overnight incubation of and saline extract of normal beef lung. Subsequently, both tubes at 0-5° and centrifugation at 0°. dipalmitoyl lecithin has been considered by most in- Lysolecithin was prepared by the method of LONG vestigators to be the principal surface active phospho- and PENNY [15], using 0.5-2 mg purified lecithin emul- lipid in mammalian lung. Since lecithins with specific sified under nitrogen in 2 ml 0.01 M deaerated CaCl2 fatty acid components in general cannot be selectively solution, pH 6.8, to which was added 1 mg of dried separated from total lecithin by present methods, the Naja naja venom [18]. The solution was overlaid with evidence for the presence of dipalmitoyl lecithin comes 10-15 ml redistilled diethyl ether and nitrogen and from the high percentage of palmitic acid found in sealed; the reaction mixture was stirred constantly at lecithin isolated from lung [4, 5, 7, 17] and from isola- room temperature to completeness at 4 h when lyso- tion of dipalmitoyl lecithin in alveolar washings from lecithin appeared as a thick colorless gelatinous preci- rabbit lung [2]. pitate. The free fatty acids split from the /?-carbon The most adequate characterization of the fatty dissolved in the ether phase. The lysolecithin was acids esters of lecithin currently requires that those on washed six times with ether and the washings added the ^-carbon be examined separately from those on to the previously removed ether layer. the a-carbon. The finding that the acetone-precipitat- The optimal time required by phospholipase A ed fraction contains the surface active lung lecithin, (EC. 3.1.1.4) (Naja naja venom) to split the /S-carbon suggested that this fraction in particular be investigat- fatty acid ester bond of lecithin was established by ed during fetal maturation. This is so because surface sampling reaction mixtures of varying concentrations activity of lung appears as a phenomenon of late fetal of venom and lecithin at 0.5-h intervals. The samples development and increases after breathing. It was were extracted from the reaction mixtures with me- thought that these studies might provide basic informa- thanol and chloroform and applied to thin layers of tion about possible specific fatty acids patterns in leci- silica gel H. The reaction was completed (no un- thin structure associated with the development of cleaved lecithin remaining) after about 3.5 h depend- surface activity in fetal lung. ing upon the total amounts of lecithin used initially. This is a report of quantitative studies with gas-liquid In the approximate proportions of venom to lecithin chromatography (GLC) of the fatty acids on the 10% of total mixture) The isothermal column temperature was 180°, the in- and less than 5% for minor mixtures (< 10% of total mixture). Isolation and verification of PDME from lung and liver were done as previously described [10]. Surface activity measurements were made in standard fashion on a modified Wilhelmy balance, as previously described [8]. On compression of the surface, FA minimum surface tension below 15 dynes/cm for three consecutive cycles was considered 'normal surface activity'.

Results

Total Fatty Acids in Lecithin Gross measurements of the total fatty acids of lecithin are shown in table I. No clear tendency toward a greater percentage of saturated fatty acids as gestation pro- gressed was found in whole lung lecithin during representative stages of development; only a slight increase in percentage of palmitic acid was seen. In the lecithin from alveolar wash, there was an appreciable increase in percentage of palmitic acid with fetal development, but no differences were seen in the percentages of saturated fatty acids among the stages compared. The total fatty acids from alveolar wash lecithin, however, showed a higher percentage of saturated fatty acids than were found in whole lung lecithin, confirm- ing the findings of MORGAN et al. [ 17] on adult dog lung.

Comparison of Fatty Acids in Acetone-soluble and Acetone- precipitable Lecithins Fig. 1. A representative sampling run on a thin layer Table II shows representative examples of consti- plate of the lecithin/cobra venom reaction mixture tuent fatty acids on the a- and /S-carbons of three groups after 4-h incubation is shown on the right. IX: lyso- of lecithins: 1]) surface active acetone precipitable; lecithin produced. FA: ^-carbon fatty acids split from 2) nonsurface active acetone soluble; and 3) nonsur- the lecithin (L) isolated from lung. Approximately 20 face active acetone precipitable. The latter group are fig lecithin were applied on silica gel H and chromato- lecithins from liver and kidney which, although insol- graphed in chloroform-methanol-water (65:25:4), uble in cold acetone, are not surface active (do not with charring at 280° after 50 % H2SO4 spraying. reduce the tension below 15 dynes/cm on compression Biochemical development of surface activity in mammalian lung 355 of the surface in a Wilhelmy balance), while the first sented in unified fashion to show the quantification of two groups are from lung. the various forms of lecithin. The table presents data Evident is an extraordinarily high percentage of showing the rising concentration of acetone-precipi- saturated fatty acids on both a- and jS-carbons of the table lecithin in whole lung to a peak with breathing. surface active lecithins. By contrast, the acetone- The most striking finding was the marked increase in soluble lecithins had predominantly unsaturated fatty acetone-precipitable lecithin in alveolar wash after 1 h acids on the /S-carbon, although the a-carbon fatty acids of breathing. resemble those of the surface active lecithin. A very high percentage of saturated fatty acids on the a-carbon Fatty Acids of Alveolar Wash Lecithin: Acetone Precipitated and a predominance of unsaturated fatty acids on the The fatty acid components on the a- and /S-carbons j8-carbon were found in the acetone-precipitated leci- of acetone-precipitated lecithin from alveolar wash thin from adult rabbit liver. The adult kidney lecithin during gestation are shown in table IV. Both the a- and had a high percentage of unsaturated ^-carbon fatty yS-component palmitic acid (Gi6:o) rose abruptly after acids but had less than 50% saturated fatty acids on day 29, when alveolar lecithin becomes surface active the a-carbon. [8]. The greatest increase in a- and (S-palmitic acid In general, the principal differences between the followed the onset of breathing. These changes are two groups of nonsurface active lecithins include a plotted in figure 2. significantly greater percentage of palmitoleic acid Other fatty acids also varied during development (Ci6:1) in the acetone-soluble lecithins on both a- and (table IV), but only the palmitic acid changes corre- /3-carbons while the lecithins from liver and kidney lated with surface activity in alveolar wash lecithin. had greater concentrations of oleic acid (Ci8:o) and arachidonic acid (C20:4). Fatty Acids of Alveolar Wash Lecithin: Acetone Soluble Table V shows the percentage composition during Quantitative Measurement of Lecithin in the Lung of the fetal development of a- and ;S-carbon fatty acids in Developing Rabbit Fetus, the Newborn, and the Adult the nonsurface active acetone-soluble lecithin from Although much of the data in table III has been alveolar wash. Palmitic acid was the principal a-carbon presented elsewhere [8, 10, 12], it has not been pre- fatty acid, increasing markedly from day 29. By com-

Table I. Percentage composition of total fatty acids in whole lung and alveolar wash lecithin in rabbits

1 Fatty acids Whole lung lecithin, ' Alveolar wash lecithin, % Fetus, Fetus, Adult Fetus, Fetus, Adult 23 day 31 day rabbit 23 day 31 day rabbit (term) (term)

Ci4:o, myristic acid 2.2 4.2 1.6 6.9 3.7 2.2 Gl4:l 0.5 1.0 0.6 3.7 1.3 0.1 Gl4:2 0.7 0.4 0.3 - - 0.7 Gi6:05 palmitic acid 45.3 48.0 49.8 46.7 53.8 60.0 Gi6:1, palmitoleic acid 6.3 12.5 6.7 7.2 11.6 8.3 Cl6:2 - 0.4 - - - - Ci8:0, stearic acid 3.9 5.1 5.1 11.5 5.8 3.4 Gi8:i, oleic acid 19.3 12.5 15.1 18.6 12.7 13.0 Gi8:2> linoleic acid 9.9 9.5 15.9 5.5 6.8 10.5 Gi8:3, linolenic acid 0.2 0.7 1.5 - 0.6 1.0 C20-.0, arachidic acid 0.2 0.2 0.1 - 0.3 - C20:4, arachidonic acid 11.3 4.8 3.0 - 3.2 0.5 C22-.0, behenic acid _ 0.4 0.3 _ _ — Saturated fatty acids 51.6 57.9 56.9 65.1 63.6 65.6 Unsaturated fatty acids 48.2 41.8 41.3 35.0 36.2 34.1

1 The convention for identifying fatty acids includes the carbon chain length and, following the colon, the number of unsaturated double bonds. 356 GLUCK, LANDOWNE and KULOVICH

parison, there was an appreciably smaller percentage 7 9 3 6 3 8 2 6 in of palmitic acid in the /S-carbon fatty acids of acetone-

"3 0. o CM 0. 1 10. 74. 25. "ft 3 22. 20. 42. soluble lecithin. There was a relatively high percentage of /S-carbon myristic acid between days 25 and 29. The percentage compositions during late gestation of CM CO CO CT) CM CO a- and /J-carbon palmitic acid in the acetone-soluble CO CO CO i CO m 4 CM CM o 5 i—i lecithin from alveolar wash of the fetus are plotted in I 3 figure 3.

in m CTl CM to Fatty Acids of Lecithin from Residual Lung Parenchyma after 5 .> CM co CM CO CO 1 CO CO CO "ft" m CM Alveolar Wash: Acetone Precipitated d 5? This fraction representing the surface active paren- ti a2 -a II chymal or 'intracellular' lecithin showed consistently ft g co CM CO CO o high concentrations of palmitic acid in both a- and 1) o CO co in CO 1 1 CM CM CO in CO /5-carbon fatty acids in the fetal state (table VI). After CM si 6 h of breathing by the newborn rabbit, however, there CJ CtJ was a 50 % drop in concentration of palmitic acid on CO CO CT)CM CO 3 o o the a-carbon of the isolated leicthin; this was seen also 4 CJ CM CM CM 1 1 5

1 o CM o CO 2 K in the 2-day-old rabbit where it was most marked in U "5 *"•!-• CM 3 cti CJ -ci .t^ the /S-carbon palmitic acid. Lecithin from adult rabbit is g S3 gdS had low fi- and high a-carbon palmitic acid. Figure 4 o CO o in CM CO CM o co illustrates the percentage composition of the major

CO CO CO 1 CM o 9 2 fatty acid, palmitic acid, on both a- and jS-carbons of 1-8 3 in O m the surface active acetone-precipitated lecithin from residual lung parenchyma after alveolar wash in the

4 m in O CM CTl 0 if oq 9 developing rabbit fetus and after birth. 1. CO 0. 8. c-> CM 1 to rM cd 1 $• o . Fatty Acids of Lecithin from Residual Lung Parenchyma after kg I3 8"to ?'O Alveolar Wash: Acetone Soluble r^. co in CT) ^ ^o Acetone-soluble lecithin from residual lung paren- d T3 > s £ in r--CT) C M o co tO CO tion between nonbreathing and breathing newborn - „ bo Cti and adult. In particular, postbreathing values showed U — 3 .rt CM •* CO o to co I d ii 3 r^- —^ :!J2 ftg Fig. 2. Changes in percentage concentrations of pal- *M O mitic acid (Ci6 o) during fetal development in a- and O W3 T3 : in ~H CO (S-carbons of surface active acetone-precipitated leci- 11 CO CM CM O CO CO d CM r^ thin isolated from alveolar wash. The percentages CJ t> •3-11 CO —t T3 ° plotted actually are percentages of palmitic acid in --H CtJ either the a- or /S-carbon fatty acids, as labeled and not CO ?^* as percentages of the total fatty acids on the lecithin CT) m -H rt •"-• I I O molecule. The various designations (e.g., fetal states, tJ CM co ci breathing, lecithin storage in cells, etc.) refer to these experimentally determined states in the fetus and new- ct! born as previously described [8, 10, 12]. ft Fig. 3. O Changes in percentage concentrations of pal- O mitic acid (Ci6:o) during fetal development, on a- and BCtJ a u /S-carbons of nonsurface active acetone-soluble lecithin O O — O —' isolated from alveolar wash. See legend of figure 2 for TptOtOCOCOCOCOO^-'x £ cti OUUOOOOUOh explanation of terms. Biochemical development of surface activity in mammalian lung 357

Table III. Quantitative measurement of lecithin in the lung of the developing rabbit fetus, the newborn, and the adult1

Gestational age, Whole lung lecithin, Alveolar wash 1lecithin, Lecithin from residual days mg/g dry wt lung mg/:g dry wt lung lung parenchyma after alveolar wash, mg/g dry wt:lung Total Acetone Acetone Total Acetone Acetone Total Acetone Acetone precipi- soluble precipi- soluble precipi- soluble tated tated tated

Fetus 19 22.5±3.02 21 52.4±5.8 17.5s 353 1.03 0.053 0.953 50.03 133 373 22 34.4±4.6 23 37.3±5.2 8.0 29.0 1.0 0.07 0.93 35.5 9 27 24 31.6±9.1 - 25 47.2±3.6 7.5 39.0 1.1 0.08 1.02 45.8 16.7 29.1 26 50.8±1.8 27 65.5±3.2 17.0 34.5 1.2 0.1 1.1 65.2 15.0 50.2 28 76.3±4.3 18.0 50.0 1.3 0.2 1.2 74.9 15.0 59.9 29 80.2±0.8 28.0 48.0 4.1 0.3 3.8 76 24.0 52 30 82.3±3.5 34.0 49.0 4.1 0.4 3.7 78.2 30.0 48.2 31 84.2±1.9 43.0 40.0 4.3 0.35 3.9 79.9 44.0 35.9 Newborn 1 h breath 84.0±2.1 43.8 41.2 19 9.8 9.0 64.6 34.0 32.0 6 h breath 71.8± 36.0 35 11 5.0 6.0 60.8 31 29 2 day 68 29.6 38.4 8 3.6 4.4 60 26 34 Adult 58.0±l.l 28.0 32.0 6 3.4 2.6 52 28 24

1 Mean measurements from 22-46 fetuses for every value shown. 2 Standard deviations are shown for total lung lecithin to indicate the spread of measurements. 3 Only the means are shown for the other values since they represent fractions of the whole lung lecithin.

100-i 100- \°— Fetal state —* Breathing K~ Fetal state —• Breathing 90- Lecithin storage in ' ,6h 90- Lecithin storage in ^ 6h cells cells / Alveolar lecithin • ^* Alveolar lecithin 80- becomes surface ) r 80- becomes surface active / ^a-16:0 active 70- / 70- Y (\ N / A 60 - /°" 60- f f a-16:0 50- ^ (3-16:0 50- in / 3 / to 40- >». a) & 40- J • < \ CXJ1- *• S 30- S 30- o. v"\ /13-16:0 42 .-<• £ 20- +°- on20 - )*' 4— , / c O o c ^ 10 - ^ 10-

23 24 25 26 27 28 29 30 3 6h 2d Ad 23 24 25 26 27 28 29 30 3 6h 2d Ad Days of gestation Days of gestation Fig. 2. Fig. 3. 358 GLUCK, LANDOWNE and KULOVIGH

CM CD in CM CO

to . co . 100- CD •* 0 CO to 1 d 1 0 74 s ?5 |^- .Fetalslate —• BreatHng "0 90- Lecithin storage in ^ 6h CO co 0 CD in CM CN cells a m CO CO CM CO O I d 1 1 CM CO CD ^ Alveolar lecithin 80- becomes surface "1 in 0 co m CM CO CM active 1 co CM CM 0 to CO 1 d 1 O CM 70- -* t CM a to \ -0 R-1&0 CM CM in in in CD O q 60- / —-< A r < 8 CD •-< *—* ~ 1 O 1 1 cd 3 CD > 50- / S CM CM in co CD to CO CN s c \ T\ (3 ^C CO CO to ^— I d 1 d cc id tfa IS >—1 in '—1 CM 8 40- \ \ •

° n CD 0 to CM CD CD in S-16: 8 CD CO CM rt O I d 1 1 CO to 5 30- scithi

X) co CO CO CD CM CD ^ 20- b «Q. CD in CO , 1 1 d 1 d to r^ CM •s ( CO 5= 10- s O 0 m CO CM to CD CO 8 —• CM CD to m 1 —' 1 1 CO a 23 24 25 26 27 28 29 30 31 6h 2d Ad CO CM ^h CO CD CM in i Days of gestation =CL co CO m 1 1 CM g CM 0 ° CO _ in 1 1 CD CO CM CD CO CO ,F%. ^. Changes in percentage concentrations of pal- ro 8 CO f to in to CO 1 ,—^ 1 1 CO to 05 <£> CO •—< mitic acid (Ci6 o) during fetal development, on a- and c : CO CM m r-- m CM co to CM to i8-carbons of surface active acetone-precipitated leci- l «=L CD in CO CO 0 1 1 CO $ CO CO thin isolated from residual lung parenchyma after CD -car CM alveolar wash. See legend of figure 2 for explanation rt 0 in co t~. CO CM c/T 8 O m to , , 1 1 | ^ cd of terms. 3 1—' 1—' '—' •—' 1 CO »—' cS 8 O to CM —1 m CO co —1 j CQ. •"^ to 1 —| CM 1 cd to O in 2 to CO CO ? 100- •3 CM \— Fetal state -• Breathing O CM CO 0 CO CD q 8 CO CO to 1 1 1 CM Lecithin storage in " 6h co 90- • /

15 60- CO 58 s; A X ^_ 0 i CD 0 CO CO CO in < 0 it m . TO . u, 50- OQ. CD CM CO co to 1 1

19 .•2 1 m 58 0 "> 40 - CM 1 0 1 CO in I— '—• CO CM ^^ co \ / 0-1 8 0 CO CO CD to 0 1 C^ 1 1 0 CD •—1 CO I—1 t—1 to CO 2 30- i 0B O CM CM 01 CO CO in to CO to c ea CD 1 CO 1 d t 20" r £^ 2 to 0 I CO '"'6 b CM ^ 10- CM CM CO in CD 0 . . R-16:0 8 CD CO 0 '-H cd 1 1 1 44 28

CD CO CO CM CD CM 0 CD CD 23 2425 26 27 28 29 30 31 6h 2dAd CQ. ,—1 CM CM CM 1 1 1 CO 1 d Days of gestation r^ —' co -^ CM 00 CM CO m m r^> cq CO 8 CD CO CD ,—1 1 d 1 1 in Fig. 5. Changes in percentage concentrations of pal- to TT °° mitic acid (Ci6:o) during fetal development, on a- and

a 1 "" /S-carbons of nonsurface active acetone-soluble lecithin ur 0 0 —< 0 "< C4 CO 0 0 r3 d isolated from residual lung parenchyma after alveolar •5 S 00 CO CO 0 Si 0 P cN ai wash. See legend of figure 2 for explanation of terms. Table V. Percentage composition of fatty acids esterified on a- and /?-carbons of acetone-soluble lecithin from alveolar wash Fatty Gestational age of nonbreathing fetus, day Newborn, 2 day Adult acid after 6 h 21 23 25 27 28 29 30 31 breathing a p p a (3 a P a p a p P a p p P P

Cl4:0 1.5 2.1 3.8 2.4 2.5 53.4 4.5 59.0 4.5 20.9 1.1 54.7 2.3 2.8 4.1 18.6 6.1 4.5 5.7 1.4 1.8 4.2 to 48 9 31.0 37.3 15 3 36 7 15.1 S4 H 14 8 40.7 29.5 65 6 16 9 69 0 35.6 61 1 23 3 69.8 26.1 77 6 23 8 68.8 18.6 o5 5 0 09 10 2 7 9 9 5 6.2 16 0 7 7 14.0 11.2 7 3 8 0 8.7 18.8 9 16 2 11.1 29 6 9 5 20 0 6.9 17 7 13.7 2.1 19.4 7.3 13.7 2.2 11 q 4.2 11.4 6.6 11.4 2.0 8.7 2.5 5. 1 1.7 4.8 0.6 2.9 1.7 5.3 0.4 3 Cl8:l 22.6 24.4 24.1 18.3 17.2 9.3 21. 3 8.1 18.9 15.0 9.7 7.7 8.0 18.6 8.1 9.9 6.1 12.5 0.4 18.9 13.0 24.2 S Cl8:2 5.4 15.8 3.0 16.3 20.3 8.0 11.2 6.0 10.3 10.9 4.9 7.7 3.2 18.0 6..8 11.1 2.0 22.3 3.6 30.5 3.5 31.3 Cl8:3 1.2 6.5 2.0 13.9 - - _ _ - 1.7 - _ 0.6 - 7.3 - 1.8 _ 2.5 0.6 2.7 $ 1 P. O 7 Q ______17.1 18.4 5.7 3.9 2.9 2.9 3.7 2.4 1.0 0.7 B Total o> Saturated, 0/ (S5.7 35.2 60.5 25.0 52.9 70.7 51 0 78.0 56.6 57.0 78.1 73 6 80.0 40.9 75.6 51.5 80.7 31.2 86.2 26.9 75.9 23.2

Unsaturated, •1 0/ 34.2 64.7 39.3 74.8 47.0 29.2 48.5 15.8 43.2 42.7 21.9 26.3 19.9 58.9 24.2 48.2 19.2 68.6 13.5 72.9 24.0 76.6 /o

o Table VI. Percentage composition of fatty acids esterified on a- and /3-carbons of acetone-precipitated lecithin from residual lung parenchyma after alveolar wash

to 8. o ! 14. 17. 10. 78. 40. 21. position changes in a- and /3-carbon palmitic acid in 3 ^_, _ the nonsurface active acetone-soluble lecithin from < o to •* to o to to —' CO •* m o o CM CO CO residual lung parenchyma after alveolar wash. $ CO

CO CO CD o CD CM CO . . Fatty Acids of Lecithin Synthesized De Novo by the Breathing CM CM O o CM 1 1 in 14 CM CO 74 CM CM Fetus -o CM © CO CD in CM CO CM © CO The rabbit fetus delivered by cesarean section after 1 H CO CO —' co I ^ CM O 9

in 2 28 full days of gestation synthesized de novo 100% of

_ t^ his acetone-precipitated surface active alveolar wash g C O 60 co CO CM rt m rt CO to o CM 1 o CO to lecithin after 1 h of breathing [10]. The fatty acids 2 o CM r- I CO from this lecithin are shown in table VIII and were CD CO m to in CM in CD CO m predominantly a-palmitic//S-palmitic. I •H io (D 'CD iO CO

CO CM "^ CM lO OI Intermediate Compounds in the Biosynthesis of Lecithin by the •* O I O CO O | ^ —« co to co Methylation of Phosphatidyl Ethanolamine Of the two principal known pathways for de novo •* co ai 05 in to i biosynthesis of lecithin in the lung of the rabbit [9, 10], the trimethylation of phosphatidyl ethanolamine (PE) o •—' —< CO m CD co o has identifiable intermediate compounds, phosphati- CM CD —i O 1 -I m CM CM co o in CM $ 13 _ t^ cn o CO co CD CD Table VIII. Percentage composition of fatty acid esters CM to co to o o 1 CM CM CO CQ. 4 o 4 CO td co after 1 h of breathing

2 2 CM CD co to CM •* CM CD co to •* CO Fatty acid a /S --H r^. Tf co m m csi r-^ r~ CM o 12.6 10.5 Cl6:0 69.7 61.2 Cl6:l 4.4 4.0 6.8 1.9 "3 Gl8:0 § Cl8:l 4.2 10.0 8 to 05 t-; in to o to CD Cl8:2 2.3 8.9 C oi N 6 ^ d in 0 CO - 1.6 o CM -H rt CM t-H C20:0 -d CM C20:4 _ 1.8 © CD © CO CD co CM to tc m O to o td co % CM CM "S to co CD o to m CO tO CM 3 O CO 1 m I I CD in •* •3 2 CM 2 a — Palmitic a. — Palmitic m acid acid CM CO CD o to CD to O « CO CM CD to I in * (16:0) (16:0) m —< —• , to co Palmitic Myristic acid — fi acid — R (16:0) (14:0)

I - P0« Choline — PQ4 Choline

| Lecithin synthesized by Lecithin synthesized choline incorporation by methylation 8 pathway pathway

Fig. 6. Suggested schematic structure of surface active o o — o lecithin synthesized by each of the two major pathways CO CD CO CO CO CO OOOUUOOOOh for the biosynthesis of lecithin. Biochemical development of surface activity in mammalian lung 361

dyl methylethanolamine (PME) and phosphatidyl di- tO CO LO "^ LO t— tO I CM CM CM ^ -H 1 | | methylethanolamine (PDME). The PME and PDME are not found in significantly measurable quantities y in alveolar wash of rabbit (and sheep) fetuses and new- borns, and then not until after several days of life. Table IX compares the fatty acid components of co o co co o CO CO —• CO the acetone-precipitated fractions of adult rabbit PE, PME, and PDME from lung as well as PDME from adult rabbit liver. There was much variation in PE co to N r^ o GI io co ^H cj; in fatty acid composition from animal to animal; CT> I O CM « O CO © 1O CM r-^ LO —i CM PDME showed little individual variation. Because of the variability, PE isolated from 12 lungs of adult rabbits was pooled and the fatty acids compared with tO LO those of PME and PDME. LO I CM tO CO —i to -H |CM —I CO LO —i -H As seen in table IX, PME and PDME resembled each other in total fatty acid composition while PE was considerably different. In the comparison of the ~-> CTi CO O CO « .

. a- and jS-carbon fatty acids in the acetone-precipitated . CM CN t^ 1 CM CO . 10 56 72 27 fractions, the ^-carbon fatty acids of all three compounds were similar. The oc-carbon fatty acids of PE differed markedly from those of PE or PDME, while CD LO LO CO CM those of the latter two resembled each other. . « ! . 58 41 The a- and jS-carbons of acetone-precipitated sur- I face active PDME from adult rabbit alveolar wash at showed largely on a-palmitic acid/jS-myristic acid G.

CM 4 o distribution. The ^-carbon fatty acids from liver 8 CO 1 CO CO CO NfJd 1 4. 00 PDME were similar to those of PDME from lung whereas the oc-carbon fatty acids were considerably different. Liver PDME was not surface active [8]. qioiDcq-jcoiooiiooi ^hcoa^^^cTJcdco^HCJicM o ai -* : —' CM Discussion

MORGAN et al. [17] first established that there is a t—t I IOIO-HOLOOO-HCM CO ~H CO CM ^H r-1 ^H significant difference between the phospholipid com- Q ,3 to co 3 a PH position of whole lung homogenates and that of al- * p I veolar wash of adult dog lung. OH The synthesis of phospholipids in lungs of fetal •* to s I a> to co lambs was studied by GHIDA et al. [4] who reported •a •£•'.&• increasing amounts of phospholipids in lung with ad- vancing gestation, similar to findings reported in rab- W cf COOr^tDCMCOCOCOCOCO bits [10], with the greatest increases found in the leci- II ojco'^coinoirtindB) PH 3 i—i T—i CM *—< thin fraction. BRUMLEY et al. [3] who also studied fetal lambs, found increasing phospholipid concentrations in the lungs after 120 days gestation as well as increasing concentrations of 'disaturated phosphatidyl choline'. These workers, however, did not examine the lecithins in alveolar wash, their studies having been done on homogenates of whole lung. Surface active lecithin can be separated from the total lecithin fraction by acetone precipitation [10]. The fatty acid ester components of acetone-soluble O O O -^ O —i =o o * -3 t; a and acetone-precipitate surface active and nonsurface active lecithins were compared in that report. No sur- 362 GLUCK, LANDOWNE and KULOVICH face active acetone-soluble lecithin has as yet been The lack of increase in /S-carbon palmitic acid in identified in this laboratory, although some acetone- lecithin from alveolar wash and the lack of a concurrent precipitated lecithins are not surface active. Precipita- drop in percentage of /?-carbon palmitic acid from tion in cold acetone appears to be a property less easy residual lung lecithin to match the changes seen in the to define than the property of surface activity. In corresponding a-carbon palmitic acid of the lecithin general, surface activity appears to be associated with indicated that significant percentages of fatty acids a high percentage of saturated /S-carbon fatty acids as other than palmitic acid were contained on the /S-car- well as saturated oc-carbon fatty acids on the lecithin. bon of the lecithin which passed into the alveolar wash Acetone-soluble lecithins (nonsurface active) may have from the cells. The principal fatty acid probably was a high percentage of saturated a-carbon fatty acids but myristic acid (Ci4:o) which rose in the alveolar wash the percentage of saturated ^-carbon fatty acids is low. lecithin fraction to about 17 % of the /S-carbon fatty The nonsurface active but acetone-precipitated leci- acids. thins may also have a low percentage of saturated fatty By day 2 a sort of 'steady state' was reached when acids on the /S-carbon and highly saturated fatty acids lecithin compositions became similar to those found on the a-carbon, which probably accounts for their in the adult rabbit. Alveolar wash lecithin from 2-day- being nonsurface active. They differ, however, from old rabbits, when compared with alveolar wash lecithin the acetone-soluble group particularly in a near lack of the 6-h-old newborn, showed a slight increase of of palmitoleic acid (Ci6:1) on both a- and /S-carbons /S-carbon palmitic acid as well as a sharp drop in the compared with the acetone-soluble lecithins; in addi- /S-carbon palmitic acid of lecithin from residual lung tion, they have somewhat greater components of oleic after alveolar wash. In the adult rabbit, /S-carbon pal- acid (Ci8:1) and arachidonic acid (C20:4) on both car- mitic acid on the surface active lecithin from residual bons. lung comprised a small percentage of fatty acids, while The data in this report show that the appearance percentages of both a- and /S-carbon palmitic acid late in gestation of normal surface activity in the alveo- were extraordinarily high in the acetone-precipitated lar wash lecithin from nonbreathed fetal rabbit lung surface active alveolar wash lecithin in the 2-day-old is characterized by marked increases in the percentage and adult rabbits. of palmitic acid (Gi6:o) esters on both the a- and /S- These findings, together with those previously re- carbons of the surface active lecithin. ported of the localization [8, 10, 12] and de novo bio- From day 25 of gestation till term, the rabbit stores synthesis of lecithin [10] and the quantitative summary increasing concentrations of lecithin intracellularly in in table III in the present report, show that the rabbit lung [8, 10, 12]. In nonbreathed fetal lungs during this fetus approaching term stores large quantities of sur- period, surface active acetone-precipitated lecithin face active dipalmitoyl lecithin as well as other leci- isolated from residual lung parenchyma after alveolar thins. This fraction probably includes some palmitoyl- wash also showed consistently high percentages of myristoyl lecithins which are released rapidly to the palmitic acid on both a- and /S-carbons. alveolar surface after the onset of breathing. By day 2 Even at term, only about 11 % of alveolar wash of life, the 'steady state', there are relatively few differ- lecithin is acetone-precipitated surface active com- ences between lecithin from alveolar wash and from pound [8, 10, 12]. Within an hour after the onset of residual parenchyma after wash in the fatty acids of breathing, this increases to 50% of the lecithin in al- the 2-day-old rabbit and adult rabbits. veolar wash, essentially the same proportion found The acetone-soluble lecithin fractions, in alveolar throughout the subsequent life of the rabbit. After wash and in residual parenchyma after alveolar wash, breathing, however, the fatty acid ester patterns of the were not surface active [8]. In both fractions the a- lecithins changed markedly from those isolated from carbon palmitic acid was the most abundant fatty acid nonbreathed fetal lung. found throughout the gestation period. The /S-carbon In the full-term rabbit after 6 h of breathing, the palmitic acid, however, although a major component, percentage of palmitic acid on the a-carbon of the was always less than 35% and usually less than 25% surface active acetone-precipitated alveolar wash of the fatty acids of the /?-carbon. Myristic acid (Ci4:o) lecithin had increased to approximately 85% of all throughout gestation comprised a significant propor- fatty acids esterified on that carbon, but there was no tion of/J-carbon fatty acids of the acetone-soluble leci- corresponding increase of palmitic acid on the /S-car- thin. bon. The acetone-precipitated lecithin from residual There were high percentages of oleic acid (Ci8:i)> lung parenchyma after alveolar wash showed a reci- both a- and /S-carbon components, in the surface active procal change, with a marked drop of about 50 % in fractions as well as in the nonsurface active ones a-carbon palmitic acid and a slight decrease in that of throughout gestation. The timing and patterns of the /S-carbon. changes in this fatty acid suggested a possible precursor Biochemical development of surface activity in mammalian lung 363 role in the production of palmitic acid, perhaps by recoverable amounts in alveolar wash during the first oxidation of oleic acid, but this remains to be proved. week of life, but PDME is easily isolated from alveolar Other fatty acids also showed transitory increases in wash of the adult rabbit. concentration suggesting possible special roles at par- Since PDME was found to be surface active [8, 17], ticular times in the lecithin metabolic cycle, including it was particularly important to characterize its fatty linoleic acid (Ci8:2), palmitoleic acid (Ci6:i), and acid components. If any specificity were found of arachidic acid (C2O:o)- No consistent meaningful pat- esterified fatty acids associated with PDME, this might tern, however, was obvious for these acids. The data provide a marker for assessing the contribution of the in this report do not give a clear picture of fatty acid methylation pathway in the production and localiza- synthesis or incorporation, but indicate only the fatty tion of surface active lecithin. This is true since the acid content of lecithin and other phospholipids during formation of PME was shown to be the rate limiting gestation and after breathing. step in the methylation of PE [1, 10]. Once the first At present, there are no effective technical means methyl group is attached to PE, the reaction proceeds to separate the lecithin of a specific composition from easily to completion successively forming PDME and total lecithin, nor are there any successful methods lecithin. known to synthesize specific lecithins containing non- The fatty acid composition of the esters of acetone- identical a- and ^-carbon fatty acid esters. Thus, sur- precipitated surface active PDME isolated from adult face activity of lecithin may be observed with a variety rabbit alveolar wash could not have been predicted of combinations of a- and /J-carbon fatty acids that are from the fatty acid composition of the total lecithin 'compatible' and form a compressed layer of low sur- that was isolated. Approximately 50% of the PDME face tension. a-carbon esters was palmitic acid; 60 % of the ^-carbon This study demonstrates the specificity of structure esters was myristic acid (Ci4;o) and only 10% was pal- of the actual surface active lecithin isolated from alveo- mitic acid. This is evidence suggesting a significant lar wash. It is not proper to assume, however, even content of surface active palmitoylmyristoyl PDME, from the extremely high palmitic acid content in sur- or a-16:0/^-14:0 fatty acid ester configuration. Since face active lecithin found in the 2-day-old rabbit (85 % the PDME is methylated directly to lecithin, this on the a-carbon and 62% on the jS-carbon) that this identifies another specific surface active lecithin of represents 62 % dipalmitoyl lecithin. This is the theo- known structure in addition to dipalmitoyl lecithin, retical maximum, but can range from as low as 47 % palmitoylmyristoyl lecithin. There have been no known dipalmitoyl lecithin. From day 30 of gestation through- previous reports of specificity of lecithin fatty acids out the life of the rabbit, a theoretical range between associated with the methylation reaction. 27 and 64% of the acetone-precipitated surface active These findings suggest that the /S-carbon palmitic lecithin in the alveolar wash is dipalmitoyl lecithin, or myristic acid can act as a marker to indicate the greater than any other single identifiable fraction. principal pathway of synthesis of the acetone-precipi- The major pathway for lecithin synthesis in lung tated surface active lecithin, as shown in