Phosphatidylcholine-Lysophosphatidylcholine Cycle Lysophospholipase Phosphorylcholine Cytidyl Transferase

Home , Phosphatidylcholine, Phosphorylcholine

Pediat. Res. 11: 858-861 (1977) Fetus newborn lung phosphatidylcholine-lysophosphatidylcholine cycle lysophospholipase phosphorylcholine cytidyl transferase

Phosphatidylcholine- Lysophosphatidylcholine Cycle Pathway Enzymes in Rabbit Lung. 11. Marked Differences in the Effect of ~estationalAge on Activity Compared to the CDP-Choline Pathway

FRANCIS H. C. TSA0'30' AND RICHARD D. ZACHMAN Deparltnenr of Pediatrics of rl~eUniversiry of Wisconsin, and The Wiscorlsin Perinaral Cenrer. Madison, Wisconsin, USA

Sunlmarv considered as an important factor in relation to the lung maturation. Lysophosphatidylcholine (IysoPC), a breakdown product of Lung PC is synthesized mainly from the de tlovo pathway; phosphatidylcholine (PC), might be important in pulmonary PC namely, the CDP-choline pathway (6, 9). It can also be resyn- synthesis through fatty acid exchange reactions. This study de- thesized from a PC-IysoPC cycle pathway where PC is deacyl- fines the levels of three of the enzymes of the PC-IysoPC cycle ated to form IysoPC, which is reacylated back to PC. Two pathway (lysophospholipase (LPL) (EC. 3.1.1.5), lysophospha- mechanisms for the reacylation are possible: (I) acylation with tidylcholine-lysophosphatidylcholineacyltransferase (LAT), and acyl-CoA as described by Lands (17), or (2) a transacylation of acyl-CoA lysophosphatidylcholine acyltransferase (acryl-CoA 2 molecules of IysoPC observed by Erbland and Marinetti (7). LAT) (EC. 2.3.1.23)) in developing fetal rabbit lung and com- The activities of the CDP-choline pathway enzymes at differ- pares then1 with the enzylnes of the CDP-choline synthetic ent stages of developing mammalian lung have been studied. A pathway (choline kinase (CK) (EC. 2.7.1.32), phosphorylcho- decrease of the activity of phosphorylcholine glyceride transfer- line cytidyl transferase (CyT) (EC. 2.7.7.15), and phosphoryl- ase during the devcloprnent of fetal rabbit has been reported choline glyceride transferase (PCGT) (EC. 2.7.8.2)). (13). A decrease of this enzyme activity (27) as well as choline Lung homogenates of fetal rabbits of known gestation, new- kinase activity (26) also occurs in human fetal lung at late born, and adult rabbits were used for the enzyme, protein, and gestational ages. However, conflicting results were reported in phospholipid analyses. Total lung phospholipid, PC, and pro- developing rat (10, 24) and developing lamb (4). All of these tein increased with gestational age. Thirty days' gestation, results showed an increase in the incorporation of precursors newborn, and maternal lung activities of CK, CyT, and PCGT into lung PC or an increase in the activities of this pathway had decreased to only 50% of their activities at 22-26 days' enzymes at late gestational ages or at term of these animals. gestation. In contrast, LPL and LAT activities increased 4-5- Although the PC-IysoPC cycle pathway has been suggested as fold from 22-26 days to 30 days' gestation, and increased a possible route to remodel lung PC (1-3, 11, 14-16, 23), its further in the newborn lung, finally to a level matching maternal role in remodeling lung PC to supply dipalmitoyl-PC is not well lung (about 8-10-fold higher than the 22-26 days' gestation ac- understood. Transacylation in this pathway in the developing tivities). The microsonlal acyl-CoA LAT also showed a similar lung has only recently been studied (15) by following the incor- increasing activity with gestational age. poration of IysoPC into PC using developing rabbit lung slices. In fetal rabbit lung, enzynlic activities for the apparent major The acylation of IysoPC in the developing rabbit lung micro- PC syntlletic pathway decreased. In contrast, the n~arkedin- somes was also recently reported (19). creases in LPL, LAT, and acyl-CoA LAT activities with increas- In this paper we report the effect of gestational age on the ing gestational age and at birth suggests inlportance of the PC- activities of lysophospholipase and IysoPC acylation enzymes IysoPC cycle pathway in regulating synthesis and turnover with (LAT and acyl-CoA LAT) of the PC-IysoPC cycle pathway. maturation. activities of CDP-choline pathway enzymes, and the content of Speculation phospholipids and proteins of the developing fetus, newborn, and adult rabbit lung. There are marked increases in lung IysoPC cycle pathway Our results confirm some earlier observations that the activi- enzymes with gestational and postnatal ages. This finding sug- ties of CDP-choline pathway enzymes. CK. CyT, and PCGT. gests that this cycle pathway is very important in pulnlonary decrease or remain unchanged with increasing gestational ages maturation and in the formation of surface-active dipalniitoyl- (13, 26, 27). In contrast, the activities of the PC-IysoPC cycle PC. pathway enzymes increase markedly at late gestational age. and continue to increase after birth to the adult level. Their role in relation to the maturation of rabbit lung is discussed. Study of the development of pulmonary lipids of mammalian lung showed that the concentration of total phospholipids of the hlATERIALS AND METHODS fetus increased with gestational age of all species studied (13). Among the total lipids, PC, the major lung surfactant pliospho- ANlhlALS AND PREPARATION OF LUNG HOhlOGENATE lipid, increased significantly whereas other nonsurfactant phospholipids remained unchanged at late gestational age or after A pregnant (New Zealand) rabbit of kno\vn gestation \vithin 1 term (12, 25). Therefore, the synthesis of lung PC has been hr was anesthetized \vith 10-15 mg sodiulii pentobarbital/ PC-LYSOPC CYCLE PATIIWAY ENZYhlES. I1 8.59

pourid. The fetus was removed from the amniotic sac and fetal lung, as \vcll as matcrn;~llung. was immeclintcly transferred to 0.9% saline solution chilled in ice before fetal respiratory move- ment began. Subsequently. brorichial tissuc of the pooled fetal lung ancl the rnaternal lung was removecl before tveighing. The lung tissue was slice~land re\vashcd \vith saline and water to rernovc blood. It was then homogenized in a Potter-Elvclijem homogenizer (clearance 0.005-0.007 inch) in 10 volumes of 0.25 hl sucrose with I0 strokes at high speed. All processes were carried out at 0'. The homogenate was uscd for the ass;iys of the enzynles of PC-IysoPC cycle pathway, enzymes of CIIP-cholinc pathway, and an;llyses of protein and phospholipid content. With one exception. the lungs of 3-10 fetuses of two different does at each gestational age were ;~ssayecI ;II~LI the ;~ver;ige W ul3 reported. The newborn (less than 24-hr olcl) values were the v, average of 6 pairs of lungs; 12 adults were used. hlicrosonies t- were prepared by the procedure described previously (22). All G analyses ~vcrecarried out in duplicate.

ENZYhlE, PROTEIN, AND Pl10SPI10LII'II) ANAL.YSIS The procedures for the assay of the enzymes, Cti, Cy'l', PCGT, LPL, LAT, and acyl-CoA LAT, were as clctailecl else- where (22). The phospholipids and microsornal proteins analyses have also been dcscribcd (22). The protein concentration of the lung hon~ogcnate\tans determined by the biuret nlethod (I 8) and bovine seruni albumin (Fraction V, Miles Laboratories. tiankakec, 111.) \\#asuscd as a standard. 20 22 24 26 28 30 NB A RESULTS GESTATIONAL AGE (DAYS) A: on PROTEIN AND PIIOSPIIOLIPID ANALYSES Fig. I. effect of gest:rtion:tl age the protein conccntr;~tion(e) in the rshbit lung of fetuscs. ne\vhorns (NB), and adult (A).11: effect of Protcitl. The quantity of proteiri per g \vet tissuc incrc;~sed gestational age on the concentration of total phospholipids (0).phos- with gestation;~lage and reached a n~aximurnin the newborn phatidqlcholine (a),phospIiatidyletha11c)Ian1i1ie (A), and sphingomyelin that matched the niaternal level (Fig. IA). This ne\vborn-adult (0)in thc rabbit lung of fetuses, ne\vhorns, and adult. l'he numbcrs in lung protein level (139.17 + 16.53 rng/g\vct tissuc) \vas about 3- parentheses represent litters from t\vo differcnt mothers. l'he mean fold higher than that of 20-day fetus (40.93 2 4.57 mg/g \vet values of all lungs and ?2 SD (bracketed) are shown in this and all tissue). A lag pcriod was obscrved bct\vccn 24 and 30 il;~ysof suhsecluent figures. gestation. Plros~~lrolil~itls.The concentration of total phospholipids per g tion were only 10% of the maternal activities (14.39 -t 3.73 wet tissue also increased with gestational age (Fig. In). The nmol free fatty acids (FFA)/rng protcin/min for LPL and 7.06 + most significant change was from 30 days of gcstation (13.46 + 1.75 nmol PC/rng protcin/min for LAI') (Fig. 3). LPL incre;~scd 2.29 prnol/g \vet tissue) through newborn (19.22 + 2.02 prnoI/g 3-fold from 26 days of gestation to 30 days of gcstation (1 .79 + \vet tissue) to the m:~ternal level (27.1 1 + 3.23 pmol/g wet 0.31 versus 6.62 + 0.03 nmol FFA/rng protein/min), then in- tissuc). Insignificant changes \wre observed bct\veen 24 and 30 creased further to maternal levels. LAT also increased 3-fold days of gcstation, as similarly obscrvecl for protein concentration from 26 to 30 ciays of gcstation (0.46 2 0.19 versus 1 .66 -t 0.I 1 (Fig. In). Pl~ospliatidylcholinewas about 50-60% of the total nmol I'C/mg protciri/min), irlcreasecl further in ne\vborn lung phospholipids in all developing lung and sho~vcda pattern simi- (3.09 + 1.U3 nrnol PC/mg protein/miri), and nearly doubled lar to total phospholipids. Ilorvcver, ptiosphatidyletl1:11i(~I:111linc again in n~aternallung. I'hc activity of rnicrosomnl acyl-CoA (PE) and sphingomyelin (Shl) were nearly constant before term LAT also increased wit11 gcstation;~lage as similar as LAT (Fig. and then increased to the maternal lcvcl (5.50 + 0.95 prnol/g 3.4 ). \vet tissue for PE and 3.76 t 0.75 pniol/g \vet tissuc for Shl).

ENZYhlE ACTIVITY The regulatory rnechanisni of lung PC synthesis is not kno\vn. CDP-Cholitle I'otll~~~yE~I~~~IIcs. The specific ;~ctivitiesof a11 In c~dditionto the (Ic rlo~~oCDP-cholinc path\tfc~y,thc PC-IysoPC three cnzyrnes of the CDP-cholirie patli\vay decreaseel after 26 cycle patli\vay may be an alternate crucial route of regulation. To claysoof gestation (Fig. 2). The CK specific activity at 20 days of evaluate indrrectly the improtance of cycle pathway enzymes in gestation was 3.70 2 1.59 nrnol pl~osphorylcl~c,li~ic/~~~gprotein/ rclation to lung rn;~turation,wc stuclicd LI'L, LA'I', and acyl- niin versus 2.15 + 0.75 at the maternal level; CyT was 0.25 +- CoA LAT activities in the developing rabbit lung homogenntes 0.02 nmol CDP-choline/n~gproteinlmin versus 0.13 + 0.02; and and microsornal fraction anci compared therri with ne\\,l~ornand 1'CG.I' was 0.29 nmol PC/rng protein/rnin versus 0.1 4 2 0.03. adult values. All newborn and maternal level specific activities had decreased The content of protein, total phospholipids, and PC per g \vet to about 50% of their values at 26 days of gestation. lung tissuc increascd gradually with maturation, despite an ap- At optirnurn pI1 conditions for each enzyme, the specific parent lag between 24 and 30 days of gestation (Fig. 1). AI- activity of Cti was 10-20-fold higher than the specific activities though the lag in lung phospholipid content was not ohserved by of CyT and PCGT at all gestational ages. At plI 7.4, 37", and others (13), the anlount of lung PC in 20-day fetuses and the saturating substrate concentrations, the relative rates of these adult was alnlost the same as calculated from boll1 laboratories three enzyrne activities in adult lungs were Cti > PCGT > CyT (20-day fetus; 6.1 pn~ol/gwet (13) versus 5.9 prnol/g wet (our (Table I). data) and adult: 13.5 versus 13.6). Plrospl~n~itljlclzoli,le-Lysopl~ospl~tttitlj/cI~olitre Cycle Etlzjt~les. The somewhat conflicting results of the enzyme activities of The spccific activities of LPL and LAT at 22-20 days of gesta- CDP-choline pathway in the developing mammalian lung rc- 860 TSAO AND ZACI ISlrZN

OL,I1I.i4.AdJO 20 22 24 26 28 30 NB A GESTATIONAL AGE (DAYS) Fig. 2.11: tlic cnzyrnic activity of elloline kiri;i\e (A) in the rabbit lung 0l;~~l!l+.l+II 20 22 24 26 28 30 NB A homogcn;tte of developing fetuws. ncirhorns (,\'ll).and iiilult (A). 7'111: GESTATIONAL AGE (DAYS) reaction rilixture contiiiricd 2-4 nig protein of the hc>riiogcriiite,2 111\1 hIgCI1. 1 m\1 tVl'P. 7 mi1 [~t~~~rh~~l-~~C]cht,linccliloridc (spccific activity Fig. 3. A: the enzymic activity of lysopliospliatidylcholinc-lysopl~os- 1 x 10" cprn/pniol), and 0.1 hl 'l'ris-tic1 (pl 1 8.3) in a fin:tl volume of phatidylclioline acyItr;insfcr:tsc (1-AT) (A) ;ind acyl-CoA LAT (0)in 1 .0 nil. 'I'lic rcaction w;r\ carricil out at 37' for 15 niin. 11: tlie enqnlic the r;it>bit lung Ilornogenatc arid niicrosomcs of developing fctuscs. activities of phospIior!lcliolit~c c!tiil!l trarisfcrasc (CyT) (a) and plios- nc\\borrls (,\'B),and ad~rlt(rl ). I'or LAT the rc;iction niixturc contained phorylclioline glyccritlc transfer;t\c (PCGI') (0)in the r;it>tiit lung htr 0.3-0.5 nlg protcin of the hornogcnate, 0.4 mhl I-[I-"C]palnlitoyl-2- niogcnatc of tlcvclopirig fetuses, ne\vborris, ;rncl adult. For CyT, tlle lysopt1ospll;itidyIcI1oline (specific activity 1 x 1 OQpm/prnol). 0.15 hl reaction riiisture cont;ii~icd2-4 nig protein of the homogcriatc. 1 m\l [I , NaCI, arid 0.1 hl Tris-IICI (pll 8.0). The rcaction was c;~rric~lout at 37" 7-14C]plio\phorllcholirlc(specific ;tctivity 3 x lo" cpm pniol), 1 .5 rn\l for 5 niin. I-or ac!l-CoA LAT. the reaction \\as ;it pH 7.4 ;in11 contained \lgCI, :inil CTP, and 0.1 hI Tris-succinate (pll 6.8) in a fin;il volutnc of 0.2 nig rnicrosonial proteins ariil 0.1 rnhl palmito)l-CoA; tlic rest of tlie 1 .0 ml. For PCGT, the rcaction rliixture contained 2-4 mg protcin of the conditions acre the s;tnlc as for LAT. hlicrosomcs of 28-day and 30-day honiogcn:~tc,50 riihl hlgCt,. 0.44 1nh1 CI)I'-[I .2-"C]choli~ic (spccific fctuscs ant1 the :itlult wcrc prcp;~rcilfrom 12 litters, 7 lit~crs,and 1 activity 3 x IOkpm/pniol), and 0.14 hl .fri\-IICI (pll 8.3) in a final adult, rcspcctively. II: tlie enryrnic activity of Iysophospholip3se (LI'L) voluriic of 1 .7 ml. 130th reaction\ \\ere carricil out at 37" for 15 niin. (a) in tllc rabbit lung 1lornogcn;itc of developing fctuscs, nc\rhorns, ancl adult. T11c rcaction conilitions for LI'L were tlie same as for LAT. Tahlc 1 . Sl)c,r.ijic.crc~ti,~itic,.s of cllolitrcp~I'IINs(' (C'K), ~)lro.splrorjl(~I~oIit~c~c~j~ticl~ltriir~s/;.r(i.rc~ (CJT), rrtltl cholinc p;rtl~\\ay);llso ilccrcaceJ \\.it11 incrc;r\ing gestational age plro.vl)l~orjl(~I~oIi~~(~glj.c.c~ritlc~ trritr.sfirii.sc~ (I'C'GT) (11 l)/l 7.1 it] irl clcvcloping rabbit lung (Fig. 711). If content of protein is (i(l11lt corrcctcil for. the ilccrcasc of the cnzynie specific activity may he . .- ... ~~ -- - clue to an increase of the protein concentr;ition. Rece~itly.Koo- Sl)cciI'ic activity'. (nn~olcproduct2/mg ncy ct (I(. (1 9) also ohscr\~etla tlccreasc of tlic spccific activity of I:nryn~c protcin/min) X I0 ------.- - - ~ ~ -- CK, PCG'I'. and Cy71'. as \\.ell a\ gl!ceropllosphatc pliosphati~l!I- CK 37.30 2 6.07 transfcrasc (EC. 7.7.8.5) \\it11 increasing gcstatiorlal age. except CyT 0.53 -t 0.02 tliat they found increasing PCGT alid CyT activities in the I'CGT I .SO t 0.02 nc\vhor~ilung \vhicli \vcrc not obscrvcil in our study 2nd tliosc' of ~ others (2 1). I h.le:in v:ilucs t SD. These conflicting results for CIIP-clioli~iep;~tli\\a!. cnzymcs ' TIIC prod11ct is rcl)rcscnted ac C'K. pl~osplior~lcliolinc;C'y'l'. CD1'- h;ive several possihlc csplanations. In sonic i~lst;inccs.the con- cholinc; I'CGT, pliospli;rtidylcllolinc. flicting results might I>c iluc to species iliffcrcnces in the ch:rrac- tcristics of the CII1'-cholinc path\v;iy. Anotlicr pos\ihlc explan;r- portcil from several laboratories (4. S, 10. 13. 7-1, 70. 77). tion for home discrepency is the iliffercnt cxpcrirnc~it;~Icondi- prornptccl us to iletcrniinc tlicsc enzymes in p;trallel \\it11 tlic I'C- tions used. It is ohvious 11i;rt espcri~ncnt;~lconilitions ilo Iiavc an IysoPC cycle patli\v;~y enzyrncs ill the clcvcloping r;it,hit lung effect on these cnzymcs. For cs;implc. in one report. PCG'I' 11omogcn;itc. PCG.1' slio\vcil higher spccific ;icti\,ity at early spccific activity rcnl;iiriccl colistalit in r;thl~it lung ~iiicrosonics gcstatio~l;il ages (77-20 d;iys) t11;11i late gcst;~tion;~l;igcs ;II~LI throughout gestation (71). \vllcrc:is \vith lung slices, I'CGT ac- materri;tl Icvcls (Fig. 713). \vliicli agrees \r ith previou\ ilata in tivity increased throughout gestation (15). rabbit (13) and human lung (77). CK in rabbit lung honiogenatc At opti~numpi1 for enzyme activity. \vc found that CK ha<\a ;ilso slio\vcd a dccrcasing enzyme specific activity \\.it11 i~lcreasc rcactio~lr;itc 10-fold liighcr tIi;in the rcl;itivc rates of Cy1 anil of gcst:itional ages (Fig. 24). similar to observations from the I'CGT (Fig. 2). This clid not vary in either fetal or ;ttlult rah- developing human lung (26) and dcveloping rat lung (34). 111 bit lung liomogenatcs. tlo\vcvcr, at pll 7.4 the spccific activ- adclition. CyT spccific activity (the scconil cnzynic of CDP- ity of Cy.1' \\.:IS Its? th;~nthat of I'CGT or CK (-I'ablc 1). contr;iry PC-LYSOPC CYCLE PA

to the results rcportc~lpreviously (8) \vliicli sho\veJ that rnorihcy brown adipose tissue of the rat during dcvelopmcnt. Physiol. Bohemoslov. lung PCGI' liacl tlie lo\vest rcl:~tivc activity. 17: 26 (1968). 6. Eptein, hl. F., and Farrcll, P. hl.: The cholinc incorpor;~tionpathway: In contrast to the CDP-choline patli\vny enzymes, the spccific Primary rnccti;~nism for ilc. r1oL.o lecithin syntlicsis in fetal primate lung. activities of Iysopliospholi~>:~scand ;tcyltransfcrascs of PC- Peili;~t.Res.. 9: 658 (1975). IysoPC cycle patli\\,ay incrcascrl markedly at latc gcstatiotial age 7. Erbland, J. F., and hlarinctti. G. V.: The enzymatic ac!lation and hyilrolysis ant1 after term (Fig. 3). The results sho\v an almost 70-fold of lysolecithin. Biochim. Uiophys. Acta 106: 12X (1 965). 8. Furrell. P. hl.: Regulation of pulmon:lry lecithin synthesis. 111: C. A. Villce, D. increase of the specific activity from fetuses of 24-26 d~~ysof 13. Villee, and J. Zuchcrrnnn: Re\pir;~toryI)i\tre\s Synclronle, p. 31 1 (Aca- gestational age to tlic atlult. An AI'P-Jcpcndcnt acyl:ttiori path- demic Press, New York. 1973). \v:~y \\.as also demoristratccl in tlic fctal rnicrosornnl fraction of 9. Farrcll. P. ht., and Avery, hl. E.: Ily;~linemcnlhrane di\c:~\c. Arner. Rev. rabbit lung tliat incrcascd \vitli maturation (Fig. 3A) as also ob- Rc\p. Dis. 111: 057 (1075). 10. Farrcll. P. hl.. Lundgrcn, I). W.,and Adams. A. J.: Clioline hina\c and served by liooncy cPt cil. (10). Similar increasing activities of choline phosphotransfcrasc in developing fct;11 rat lung. Uiochcm. Uiophys. LPL and microsom:~lncyl-CoA LAT \vcre also observed in tlic Res. Commun. 57: 096 (1974). developing rat lung (5). In contrast. the activity of the trans- I I. Frosolono, hl. I:., Slivha. S.. anil Ch;lrnls. U. L.: Ac)ltrnnsfcr;~\c;~ctlviticr in acylation of IysoPC (LAT) in the fctal rat lung was highcr than dog lung microsomcs. J. Lipid Res. 13: 96 (1971). 12. Cluck. L., hlotoyama, E. K., Srnit\. I{. L.. nncl Kulovich, hl. V.: I'he tliat of the adult (5). It is not kno\vn \vhcther tlicse discrepant re- hiochcrnical development of surf;lcc activity in rnarnni;~li;~nlung I. The sults of rat and rabbit tvcrc because of species tlifferenccs or es- \urfi~ceactive pho\ph(1lipid\; tlie scp;lration and distribution of surface pcrirncntal conditions. active lecithin in the lurig of the clcvcloping r:~hhitfctu\. I'ciliat. Re\. 1: 237 l'he CDP-choline path\vay cnzyrnes are certainly present in (1907). 13. Gluck. L., Srihncy, hl., and Kulovich, hl.: l'he hiochcrnic;~lileveloprncnt of the rabbit lung throughout thc span of gcst:~tional age (at least surface activity In rnan1nl;ilian lung. 11. The hio\ynthesis of pliospholipiil\ in after 22 days). Their activities ;~pp;rrcntlydccrcasc wit11 tnatura- the lung of the developing rahhit fctu\ and ncuhorn. Pcdiat. Re\. 1: 247 tion. An increase of the spccific activity of phosphatidic acid (1907). phospli(>liy~lrolasc(I'APase) (EC. 3.1 .3.1)in developing rahhit 14. ll;~ll~ii:~n,hl., and Raivio. K.: Stuilics on the hiosynthesi\ of dis:~tur;~ted lecithin of the lung: l'he irnport;~~lccof the Iysolccithin p;~th\v:~y.Pctliat. lung lias been reported (21). The authors suggested tliat tlic Rcs. 8: 874 (1974). cntlogcnous diglyccrides formed by the reaction of PAl'asc may 15. Il;~ll~nan,hl., ;~rldRaivio, K.: Form;~tionof dis;~tur;~tedlecithin through the be important in tlie regulation of PC sytithesis in the tlcvcloping lysolccithin p:ithway in the lung of the developing r;~hbit.Iliol. Neon;ltc 27: lung. Ilo\vcvcr, the specificity of fatty acids in cliglyccri~leslias 329 (1975). 16. Kyci-Aho:~ggc. K., Ruhin\tcin. D.. and Beck. J. C.: Uio\yntliesi\ of ilip;~lmi- not been studiecl for PAPasc. and PCGT in niamm;~li;tn lung toyllccithin hy the rahhit lung. C;ln;~d.J. D~ochcni.51: ISXI (1973). sho\vcd no specificity (3, 20. 23). Tlicrcfore, the I'C-IysoPC 17. L:~nil\.W. E. hl.: hlet;lholi\m of glyccrolipids. 11. 'l'he en/y~n;iticnc)l;llion of cycle patli~vaymay be a very itnportant route for tlic formation I!\olc.cithin. J. Biol. Chc~li.235: 2233 (1960). of dipr~lmitoyl-PC.7'11~ marked increase of acyl~~tionof IysoPC 18. I.;~yne.E.: Spcctrophoto~netricanil turhidimetric method\ for rnc;~\uringprc- tcins. hlethods Enzymol. 3: 450 (1957). at term and after birth indicates that this mcclianisrn is at least 19. Rooney, S. A,. Wai-Lee. .I'. S., Gohriln. L.. and hloto):~m;~,E. K.: Pho\pho- temporarily rclatetl to lung tn:tturation. Ho\vcvcr, its role in the lipid contcnt, compositio~iand biosynthesis during fetal lung developnlcnt in fortnation of lung dipalmitoyl-I'C, \vlietlier in the cytopl:~smor in the rahhit. Iliochim. lliopli!s. act;^ 431: 447 (1976). tlie endoplasmic reticulum, requires further study. A key cn- 20. Sarzal;~,hl. G., and Van Goldc, L. hl. G.: Sclcctive ut~liration of en- dogenous un\:~tur;~tedpho\pll;~tid)lcholinc :ind di;lcylglyccrolc by choline zyme in the cyclc patli\vq. pliospliolipase A,, also ncccls thor- pli~~\pliotr:~~i\Icri~scof 111011\e Iu11g ~nicroso~i~cs.Dioclli~ll. Iliopl~)\. Acta. ough investigation. 441: 423 (1070). 21. Schultz. F. hl.. Jirnencz. J. ht., hlacl)onald, P. C.. and John\ton. J. hl.: Fetal lung rnatur;~tion. I. Pho\phatidic acid pho\phoh)drol:~\c in r;lhl>it lung. CONCLUSION Gynccol. Invest. 5: 222 (1974). 22. l's:lo. F. 11. C.. and Zachmnn. R. I).: Pl~o\pli;~tid>lcholinc-l)soplio>~~Ii;~tidy!- The protein concentration, phospholipicl content. i~ntlthe spe- choline cycle p;~th~ayenzymes in rahhit lung. I. Suhcellul;~rIoc:~lir;~tion and cific activities of CIIP-cholinc patli~vayerizymes ancl I'C-IyaoI'C propertie\. I'cdl;~t. Res.. 11: 840 (1077). cyclc pathtvxy enzyriics in the developing rabbit lung have been 23. Vcrykcn, hl. hl., hlontfoori. A,. and Van Gol(lc, L. hl. G.: Some stuilics on stutlied. ?'otal lung phospholipids, PC. and protein incrcasctl the biosynthesis of the molecular species of ptiosph;~ti~l)Icholinefrom rat lung and pho\phatidylclioline ;lnil phorph;~tid)Icthanol;~~nincfro111 rat liver. with niaturation. l'l~cspecific activities of CD1'-choline enzymes Uiochirn. I3iophy\. Act3 260: 70 (1972). (CK, Cyl'. ancl I'CGI') clccrcased or remained tlic sanic \villi 24. Weinhold. P. A,, S;~n(lcrs.R.. and Stern. W.: Ilcgulation of cliolirle phos- increasing gestational ages. In contr;tst, the specific activities of phoglyceriile synthesis during lung devclopnicnt in the r;it. In: C. A. Villee, the PC-IysoPC cycle patli\vay cnzyrnes (LPL. LAI'. acyl- I). U. Villcc, and J. Zuckerrnan: Respiratory Di\trc\\ S)ndrorllc, p. 20 and (Academic Prc\s, New York. 1973). CoA LA-I') incrcascd m:trhcdly at latc gesti~tional age, ant1 25. Weinhold, P. A,. and Villee. C. A,: I'liospholipid metaholi\m in the liver and continuctl to increasc after birth to the maternal Icvcl, si~ggesting lung of rat.; iluring development. Diochc~ii.Iliophys. Act21 106: 540 (1065). the importarice of PC-IysoPC cycle p:~tli\vayin regulating syntlic- 26. Z;~chrnan.R. I).: The cnryrncs of lecithin hio\)nthcsis in Iiurn:~n ncwhorn sis and turnover \vitli maturation. lungs. I. Cholinc Linasc. Uiol. Neonate. IY: 21 1 (1971). 27. Z;~chrn;ln. R. D.: The enlymcs of lecithin hio\ynthesi\ in tiunia~incuhorn REFERENCES AND NOTES lungs. Ill. I'ho>pllor)lcllolinc gl)ccr~iIetri~~isfcrasc. I)ciIi:~t. Rcs.. 7: 632 (1973). I. Ahc, hl., Ahino, l'.. and 0111111, K.: The for~nationof lecithin from Iy\olcci- 28. l'liis rese;~rchu:~s supported hy N;~tion;llIn\titute of llc;~ltliGrant 5-ROI 111. thin in rat lung surfactant. Uiochim. Biophys. Acta, 280: 275 (1972). 17239-02 I IEU. 2. Atxl, hl., Ohno, K.. and Soto. R.: Po\\ihle identity of lysolccithin ;~c)l- 2'). We wish to tIi;~nhhls. K. Jane Ja\houshi anil hlr. Steve11 I:. Louilcn for their hydrol;l\e with 1)solecithin-lysolccithin ac)ltran\fcra\c in rat-lung solut)lc tcchnic;~l;~saistancc. The di\cus\ions with Dr. Gary R. Gutchcr throughout fr;~ction.niochirn. Hiophy\. act;^. 369: 301 (1974). this uork and Dr. John I{. Law's comment\ and suggestions on tlic rn:lnu- 3. Ahino. I... Ahe, hl., and Arai, I..: Studies on the l,iosynthct~cp;~tliwa)b of script itre gratefully ;tch~~oulc~lpc~l. molecular spccics of lccithin hy rat lung slices. Uiochim. Iliophys. act;^, 30. llcque\ts for reprints should he ;~ilclrc\\edto: I:. II. C. l's;~o.Ph.11.. Depart- 248: 274 (1971). ment of Pediatrics. University of Wisconsin, and the Wiscomin Perlnatal 4. Chld:~. N., and Ad:~rns.F. 11.: 1ncorpor:ltion of p;~lmit;~tc,glucose and choline Center, 202 S. Parh St.. hladi\on. \Vi\c. 53715 (USA). into Iccithin by fetal and ncuborn lamb lung. Pcdiat. Res., 1: 304 (1967). 31. Kcccived for puhlicat~or~Scptcnlher 23. 1970. 5. Dobia\ov:~,hl., and Ilaha. I'.: Lysophospllolipid rnctal)olisn~in lung, liver and 32. Accepted for publication Decerlihcr 14. 1976.