Pediat. Res. 10: 767-769 (1976)

Amniotic fluid fetus new born

phosphatidic acid phosphohydrolase phospholipids

Fetal Lung Maturation IV: The Release of Phosphatidic Acid Phosphohydrolase and Phospholipids into the Human Amniotic Fluid J U A N M. J I M E N E Z A N D J O H N M. JOHNSTON From the Cecil H . and Ida Green Center for Reproductive Biology Sciences, Departments of Obstetrics and Gynecology and Biochemistrv, The University of Texas Health Science Center at Dallas, Southwestern Medical School, Dallas,, U . S . A , : and The Universitv of Texas Health Science Center at Dallas, Southn~estern Medical School. Department of Obstetrics and Gytiecolog~~. 5323 Harry Hinrs Blvd.. Dallas. Texas 75235 ( U . S . A . )

Extract The relationship between surface active material and phosphatidic acid phosphohydrolase (PAPase) activity was examined in human amniotic fluid after centrifugation a t 105,000 x g for one hour. The distibution of PAPase activity between the pellet and supernatant fractions was similar to that of phospholipids in the amniotic fluid. The majority of the PAPase activity was found to be associated with the pellet fraction. Similarly, 82% of the phospholipid was also present in this fraction. Of the total palmitate present in the original sample, approximately 90'70 was found in the pellet after centrifugation. Palmitic acid constituted 75% of all fatty acids present in the phosphatidylcholine identified in the pellet. By sharp contrast only 4% of the total protein was pelletized by centrifugation. The pelletized fraction isolated from amniotic fluid after 105,000 x g centrifugation closely resembles the lamellar bodies in: (1)its phospholipid pattern; ( 2 ) palmitate concentration in phosphatidylcholine; and, (3)the presence of PAPase activity with a high specific activity. Collectively, these findings lend strong support to the concept that a lamellarlike body is released from fetal lung into the amniotic fluid. Speculation Inadequate production of surface active material by the lung plays an important role in the pathogenesis of respiratory distress syndrome of the newborn. The measurement of key enzymes responsible for the regulation of phosphatidylcholine synthesis would be useful a s another index of fetal lung maturity. The response to prenatal therapy to induce surfactant synthesis or release could be monitored by the assay of these enzymes in the amniotic fluid and help to define the normal intrauterine stimuli for the surge of phosphatidylcholine synthesis.


INTRODUCTION Several investigators have found that the maturatlon process of the fetal lung i s assoclated with an Increased production of a lipoproteln possessing surface-actlve propertles. The major phosphollpld of thls lipoproteln i s dlpalmitoyl-phosphatldylcholine (DPPC); moreover thls phosphollpld Is thought to be responsible for most of the surface-active propertles of the complex. The several enzymes that catalyze the blosynthesls of DPPC have been studled I n both adult and developlng fetal lung (3). We have found that phosphatldic acid phosphohydrolase actlvity [phosphatldate phosphohydrolase (EC. (PAPase), a key enzyme I n the regulation of phosphollpld blosynthesls (8, 17). begins to increase I n fetal rabblt lung on day 26 of gestatlon and Its actlvlty contlnues to increase and reaches adult levels Just p r i o r to parruritlon (18). I n contrast to the observed increase I n PAPase actlvlty, the activlty of the enzyme CDP-chollne: dlglycerlde choline phosphotransferase [EC. (CPTase) 1 i n lung of fetal rabblts d l d not change d u r l n g the c w r s e of gestatlon (5, 18). Tlssue speclflclty for the increase I n PAPase activlty I n the lung was suggested b y the flndlng I n l l v e r PAPase activlty d l d not Increase d u r l n g fetal development (181. We have also demonstrated PAPase activity i n human amnlotlc fluld (9). Furthermore, 4 to 5-fold hlgher activlty of PAPase was found I n the amniotlc f l u i d obtalned from pregnancies of more than 35 weeks than I n amniotlc f l u l d from pregnancies before the 32nd week of gestation. I n a study of the sequenu Qlhl 137in.amnlotlc f l u i d PAPase actlvity I n 21 gravldas i n whom repeated amnlocentesls were performed beginning at 30 weeks of gestatlon a continuing lncrease i n actlvlty was observed that reached peak actlvlty b y the 37th week. The observed Increase In amniotlc f l u l d PAPase actlvlty preceded the "surgeH of acetone precipltable phosphatldylchollne b y approximately one and one half weeks. Also, the speclflc actlvlty of PAPase i n the nasopharingeal f l u l d was found to b e 3 to 4 Umes greater than that of the amnlotic f l u l d (10). These findings support the concept that PAPase originates i n the fe:al lung. The purpose of the present study was to ascertain the realtionship between the surface-actlve materlal and PAPase activity in human amnlotlc fluld. METHODS AND MATERIALS Amnlotlc F l u l d Fractionation Twenty-five m l of amnlotlc f l u l d were collected at the tlme of cesarean sactlon from gravldas at term. To remove Intact cells and cell debrls, the samples were chilled Immediately and centrifuged at 49: at 700 x g for 5 mlnutes. The supernatant fraction of this centrifugation was removed and 1s referred to subsequently a5 the orlglnal amnlotlc fluld sample. A n allquot af the orlglnal sample was centrlfuged at 105,000 x g for 60 mlnutes at 4%. The supernatant fractlon and the pellet were analyzed separately. Gas L l q u l d Chromatography The i i p l d s were extracted from amnlotlc f l u l d b y the method descrlbed b y Folch et al., ( 4 ) . The chloroform was removed b y evaporatlon under nitrogen aT2YC. The fatty acld wmposltion of this l i p i d fractlon was determined b y procedures we have descrlbed prevlously (19). A n aliquot of the l l p l d fractlon was placed on a Slllca gel HR plate. previously activated at 100°C for 30 mlnutes. The developlng solvent was chloroform: methanol: acetlc acld: 0.9% saline (75: 25: 10: 6). The separated Ilpids were visuallred after exposure to 1 vapors. The area mlgratlng parallel to an authentic phosphatldylchollne was d r a p e d from the TLC plate. The fatty acid composltlon of the various phosphollplds was determined b y gas i i q u l d chromatography of the methyl esters of the fatty aclds (191. Each analysls was performed I n duplicate. PAPase activity was measured b y a modlflcatlon of the method of Coleman and Hubscher (9). lnorganlc phosphate was quantlfled b y the method descrlbed by Bartlett (1). T h e amnlotic f l u l d pellet fractlon was further characterlzed b y sucrose gradlent centrlfugatlon seftaratlon. I n this study, PAPase was massured by the rate of release of P u t l l l z i n g a recently developed radloactlve substrate assay procedure (20). Proteln content was measured b y the method of Lowry e l al., (11). Total phosphollplds and the phosphatldylchollne fractlons were quan?lfkl by the procedure descrlbed b y Parker and Peterson (141. RESULTS The dlstrlbutlon of the actlvlty of PAPase as well as that of the quantlty of phosphollpld, palmltate and proteln a r e presented I n Table I. The results obtained from the analyses of the supernatant fractlon, following centrlfugatlon at 105,000 x g for 60 mlnutes, and for the pellet of thls centrlfugatlon are expressed as percentages of that found I n the orlginal sample. Amnlotic flulds from slx gravldas were analyzed and the tabulated results are the average of these SIX samples. From these data I t Is apparent that most of the PAPase activlty was found p r l m a r l l y i n the 105,000 x g pellet. I n some samples. as much as 908 of the PAPase actlvity of the orlglnal sample was found I n the pellet. Slmllarly, the phospholipld was also found principally I n the pellet fraction. Of the total palmitate present I n the original sample (700 x g supernatant fraction). approximately 90% was found I n the pellet after 105,000 x g centrlfugatlon. Whlle the PAPase, phosphollplds, and paimltate content war found i n the pellet, b y sharp contrast, only a small amount of the total protein was pelletized b y 60 mlnutes of centrlfugatlon at 105,000 x 9. The phosphollpld present I n the 105,000 x g pellet fractlon was further characterlzed b y an analysls of i t s phasphollplds and their fatty acld content. The results o f these studles are presented I n Table 2 . Of the phospho; l l p l d I n the pellet, 78% was phosphatldylchollne. The phosphatldylchollne was further analyzed to ascertaln the fatty acld dlstrlbutlon. Of the total fattv aclds assoclated w l t h the ohosohatidvlchollne 80% was ~ s l m i t l cacld. he' flndlng of thls dlsproportldnateiy hlgA concentratlon of 'phosphatldylchollne together w i t h the marked enrlchment of thls fractlon w l t h paimitate strongly suggests that the material belng sedimented from amnlotlc f l u l d b y h l g h speed centrlfugatlon was slmllar I f not identical to lung surface-actlve material.

Several lines of evldence gathered from this study give strong support to the concepl that the release of the surface-active material b y the fetal lung Is assoclated with a parallel release into the amniotic fluid of one of the major enzymes involved I n the blosynthesls of phosphatidylcholine, PAPase. Moreover, the PAPase actlvlty end the surface-active material were found I n the same fractlons. I n other studles we have also shown that lamellar bodies lsolated from lung tlssue also contain a significant amount of PAPase (20) and CPTase (21). I n these stud':s I t was observed that the Specific actlvlty of PAPase I n lamellar bodles was the highest of any of the various subcellular organelles isolated from lung tissue (20). The pelletized fraction isolated from the amnlotlc f l u i d after 105,000 x g centrifugation closely resembles the lamellar bodies I n (1) Its phospholipld pattern, (2) palmitate concentration I n phosphatldylchollne; and, (3) the presence of PAPase activity of h i g h speclflc actlvlty. The density of the pellet obtalned f r m amniotic f l u i d of term gestations was also found to be similar to that of lamellar bodies isolated from lung tlssue and slmllar also to that of the surface-active materials isolated b y lung lavage. Each of these components, 1.e. lsolated lamellar bodies. surface-actlve material Isolated b y lung lavage and the 105,000 x g pellet of human amnlotlc fluld were found to contain PAPase activity of h i g h speclflc actlvlty. Collectively, these flndlngs provide strong support for the concept that a lamellar-like body Is released from the fetal lung into the amniotic fluid. The results of previous studies b y Novy, et al., (13) of the pellets obtained following centrlfugatlon of amnlotlc f l u i d ?i=oi;i term gestations monkeys also Indicate that a lamellar-llke structure to be present i n the amnlotic f l u l d of this specles. T h e chemical and enzymatic studies of human amniotic f l u i d reported herein, together w l t h the findings of Novy and colleagues, that include electron mlcrographs, constitute convlnclng evidence that the fraction obtained b y centrifugation i s of lung orlgln. Thls obtains slnce the physical and chemlcal data as well as the morphologlc studles strongly resemble those characterlstlc of lamellar bodies lsolated from lung tlssue. The finding of such a close assoclatlon of the actlvlty of PAPase w l t h both the surface-active material i n the lamellar bodies and the surface-active material found I n amniotlc fluld Is also consistent w l t h the view that these materials enter the amnlotlc fluld as an associated particle. Previously we have reported studies i n which i t was demonstrated that human amnlotlc f l u l d PAPase was of fetal lung o r i g i n (10). Novy. et al, (13) found that the lamellar bodies present i n monkey amniotic fluld w e r e of fetal lung origln. Together these flndlngs negate the posslblllty that PAPase and lamellar bodies were released into amnlotlc f l u l d from tissue sites other than the lung. These results together w l t h studles reported prevlously from our laborat o r y and others provide the foundatlon for a unifled hypothesis. Thls concept Is Illustrated graphically I n F i g u r e 1, i n which the Type II lung cell, in a s s o c l a t l ~ nw l t h the Type I pneumocyte, a r e depicted. The characteristic lamellar bodles containing the surface-actlve materlal a r e shown I n the Type I 1 cell, and are wnsldered to be blosynthetlc unlts for the synthesis of the surface-actlve materlal (20). The presence of phosphatidylglycerol and Its blosynthesls has also been demonstrated I n thls subcellular fractlon (6, 16). Previously It was wnsldered that the membrane surrounding the lamellar bodles I n the T y p e II cell was the slte of enrlched amounts of PAPase (12). However, from the results of this study we suggest that PAPase may be an Integral p a r t of the lamellar body, possibly one of the several protelns known to be assoclated w l t h the surface-actlve materlal. Thls vlew i s based on the observation that the lamellar bodles lsolated from the human amnlotic fluid contaln a signlflcant quantlty of PAPase, yet from the electron microscopic studles of Novy, g $ (13) a distinct perllamellar membrane was not discerm lbla. T h e surface-active rnaterlal syntheslzed i n the lamellar body can mlgrate to the alveolar surface, Is extruded lnto the alveolar space and thence b y fetal resplratlon reaches the amniotlc fluid. The mlgratlon of the lamellar body to the cell surface occurs via the mlcrotubule system. Failure of extruslon of surfactant was demonstrated after use of lnhibltors of microtubule formation (2). The development of respiratory dlstress I n a n adult woman, followed b y death after the Ingestion of a large dose of colchlcine t o commit sulclde suggests that a slmllar mechanism for the release of surface-actlve material may occur I n the adult human ( 7 ) . The surface-active materlal released c w l d provide the necessary coatlng of the alveolar surface to prevent collapse. Thls unlfled concept envlslons that the lamellar body of the Type II lung cell Is an organelle of blosynthetlc capability. Durlng the course of human gestatlon there Is a marked Increase i n the biosynthetic activlty of key enzymes i n phosphatldylchollne synthesls, e.g., PAPase. Thls "surge" I n actlvlty precedes the augmented synthesls of leclthin characterlstlc of fetal lung maturatlon. PAPase, which i s intimately associated w l t h the lamellar body, replete w l t h surface-active materlal reaches the alveolar surface b y the microtubular system. T h e perilamellar membrane fuses w l t h the plasma membrane of the Type II cell, a releas@of surface-actlve materlal to the alveolar space occurs and I t subsequently reaches the amnlotic f l u i d b y water movement b y fetal resplratlon I n utero. Thls would account for the higher specific found i n the nasopharyngeal f l u l d than i n amnlotlc activlty of the enzyme -e fluld. SUMMARY The dlstrlbutlon of PAPase, phosphatldylchollne, and palmltate has been measured I n human amniotlc fluld followlng centrlfugation at 105,000 x g for 60 mlnutes. Of the total PAPase actlvity I n the original sample (700 supernatant fractlon). 67% was found i n the pellet. Similarly, the phosphox llpyd was also found prlnclpally I n the pellet fractlon. Of the total palmitate present I n the orlglnal sample, approximately 90% was precipitated I n the Pellet. The flndlng of such a close assoclatlon or the actlvity of PAPase w l t h the surface-active materlal found i n the amnlotlc f l u i d i s consistent w l t h the vlew that these materlals are secreted lnto the amnlotlc f l u i d as a closely assoclated particle. REFERENCES AND NOTES

B y a sucrose gradient centrlfugatlon system. the resuspended pelletlzed material obtained from amnlotlc f l u i d layered as a band a t a denslty slmllar to that reported for both lamellar bodies (20) and surfactant (15). Furthermore, the activlty of PAPase was predomlnateiy locallzed i n thls band and the spaclflc actlvlty of PAPase I n thls fractlon was slmllar to that found i n lamellar bodies (21). These results also strongly support the view that the materlal pelletlzed b y h l g h speed centrlfugatlon of amnlotlc f l u l d was surface-active materlal


1. 2.

3. 4.

Phosphorous assay i n column chromatography. J. Biol. Bartlett, C . R.: Chem., 234: 466 (1959). Delahunty, T . J. and Johnston, J . M.: The effect of w l c h i c i n e and vlnblastlne on the release of pulmonary surface active materlal J L l p i d Res.. 17: 112 (1976). Farrell, P. M., and Avery, M. E.: Hyaline membrane disease. Am. Rev. Respir. Dls., 111: 657 (1975). Folch, J., Lees; M., and Sloane Stanley, C . H.: A simple method for





the lsolatlon and purlflcatlon o f total llplds from anlmal tlssues. J . Biol. Chem., 226: 497 (1957). Cluck, L., Srlbnev, M.. and Kulovlch, V.: The biochemical development of surface actlvli; I n mammallan lung. II. T h e biosynthesis of phosphullplds I n the lung of the developlng rabblt fetus and newborn. Pedlat. Res., 1: 247 (1967). Hallman, M., and Cluck, L.: Phosphstldyl glycerol I n lung surfactan1:l. Synthesis I n r a t lung microsomes. Biochem. Blophys. Res. Commun. .( 60. - - . 1. 1147n1 . . ., . Hill, R. N., Spragg, R. C., Wedel, M . K., and Moser, K. M.: Adult resplratory dlstress syndrome associated w l t h colchlcine Intoxlcatlon. Ann. Int. Med., 83:523 (1975). ~ L b s c h e r , C.: Clycerlde metabollsm. I n S. J . Wakll: L l p l d Metabol~sm (Academlc Press, New York, 1970). Jlmenez, J. M., Schultz, F . M., MacDonald, P. C., and Johnston, J.M.: Fetal lung maturatlon: II. Phosphatidlc acld phosphohydrolase i n human amnlotlc fluid. Cynecol. Invest., 5: 245 (1974). Jlmenez, J . M., Schultz, F. M., and Johnston, J . M.: Fetal lung maturatlon: Ill. Amnlotlc f l u l d phosphatldlc acid phosphohydrolase (PAPase) and Its relatlon to the leclthln/sphlngmyelIn ratio. Obstet. Cyneccl., 46: 588 (1975). Lowry, H. 0.. Rosenbrough, N. J., Farr, A . L., and Randall, R. J.: Proteln measurements w l t h the Folln phenol reagent. J . Blol. Chem. 193: 265 (1951). Locallzatlon of phosphatldlc acla phosphatase activlty I n Meben, C.: granular pneumonocytes. J . Cell. Blo., 53:249 (1972). Evidence for pulmonary and Novy, M. J.. Portman, 0 . W., and Bell, M.: other sources of amnlotlc f l u i d phosphollplds I n the rhesus monkey. I n C. A. Vlllee, D. B. V l l l w and J . Zuckerman: Respiratory Dlstress Syndrome (Academlc Press, New York, 1973). analvsls of ~ h o s ~ h o l l ~ l d s Parker. F.. and Peterson. N. F.: Ouantltatlve and phosphollpld fatty aclds from sllica gel thln-layer chromatograms. J. L l p l d Res., 6:455 (1965). Prultt, K. M., Charng, M. J., and Spltzer, H. L.. Physical and chemlcal characterlzatlon~ofp l g lung surfactant Ilpoproteln. Arch. Int. Med. 127.3Qll .- . . -.- 110711 \ - . . ,. Rwney, S. P . . , Page-Roberts, 8 . A,, and Motoyama, E. K.: Role of lamellar Inclusions I n surfactant productlon: studies o n phosphollpld composltlon and biosynthesis i n r a t and rabbtt lung subcellular fractions. J . L l p l d Res., 16: 418 (1975). SchacQS J., and Agranoff, B . W.: Acetylcholine stimulates hydrolysis of P -labeled phosphatldlc acld I n gulnaa pigs synaptosomes. Biochem. Blophys. Res. Commun., 50: 9311 (1973). Schultz, F. M., Jlmenez, J . M., M a c D o ~ l d . P. C., and Johnston, J . M.: Fetal lung maturallon: I. Phosphatldlc acld phosphohydrolase I n rabblt lung. Cynecol. Invest., 5: 222 (1974). Schwarz, B . E., Schultz, F. M., MacDoruld, P . C., and Johnston, J . M.: Inltlatlon of human perturltlon Ill. Fetal membrane content of prostaglandln E and F precursor. Obstet. Cynecol., 46: 564 (1975). Spltzer. H.%.. RI&, J. M., MacDonald, P. C.. and Johnston, J . M.: Phosphollpld blosynthesis In lung lamellar bodles. Blochem. Blophys. Res. Cmmun., 66:17 (1975). Spltzer, H. L., Wallls, P . A., and Johnston, J . M.: Phosphatldylchollne blosynlhesls b y Isolated lamellar bodles from lung type II cells. Fynecol. Invest., 7: 53 (1976). Spltzer, H. L., Jlmenez. J . M., and Johnston, J . M.: Perso~l Communlcatlon Supported I n p a i t b y PHS Grant 1 W 1 HD 08671-01 and USPHS Grant AM-031 08. The authors thank Mary Bob Wylle and Robert H. T r u b e y for thelr technlcal asslstance I n thls Investlgatlon and gratefully acknowledge Jaye Patton for her asslstance In the preparatlcn of the manuscript. Requesu for r e p r l n t s vhould be addressed to: Juan M. Jlmenez, M.D., T h e Unlverslly of Texas Haalth Sclence Center at Dallas, Southwestern Medlcal Schwl, D a p r t m e n t of ObsteIrlcs a n d Gynscology, 5323 Harry Hlnes Boulevard, Dallas, Texas 75235, USA. Accepted for publlcatlon May 5, 1876.




#. 17. 18. 19. 10. 21. 22. 23. 24. 25.




















A T 105,000 x g FOR 1 HOUR

Percent Phosphollpld Phosphatldylchollne


Fatty aclds present I n phosphalldylchollne 14:O


16: 0


18: 0


18: 1


18: 2


20: 4


Schematic representation of an alveolus w l t h type I and type II cells. Flgure 1 The lame'llar bodles formed I n the type II cell, after belng devoided of thelr perllamellar membrane are extruded Into the alveolar space, where they may coat the alveolar surface o r are carrled as a lamellar-like body into the amnlotlc f l u l d via the respiratory tract.

Fetal lung maturation IV: the release of phosphatidic acid phosphohydrolase and phospholipids into the human amniotic fluid.

Pediat. Res. 10: 767-769 (1976) Amniotic fluid fetus new born phosphatidic acid phosphohydrolase phospholipids Fetal Lung Maturation IV: The Releas...
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