ModifiedMethodfor Estimatingthe Phosphatidyl Choline:SphingomyelinRatioin AmnioticFluid, and Its Use in the Assessmentof Fetal Lung Maturity

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1 ModifiedMethodfor Estimtingthe Phosphtidyl Choline:SphingomyelinRtioin AmnioticFluid, nd Its Use in the Assessmentof Fetl Lung Mturity Lslo Srkoi, Hnn N. Kovcs, Howrd A. Fox, nd Thoms Kerenyi Phospholipids of mniotic fluid were extrcted, seprted by thin-lyer chromtogrphy, detected with cidified mmonium sulfte, nd chrred; the phosphtidyl choline nd sphingomyelin frctions were quntitted by trnsmission densitometry. The densities were recorded s integrtor trce units.we used commercilly vilble precoted pltes nd solvent system to eliminte the interference of phosphtidyl serine. The procedure used for visulition e iminted the irritting nd corrosive effects of the 50% sulfuric cid spry used in other methods. Omission of the cetone precipittion step decresed ssy time without loss of ccurcy. In 117 mniotic fluid smples obtined before deliveryof infntswho subsequently did not develop respirtorydistress syndrome (RDS) the geometric men density rtio of phosphtidyl choline:sphingomyelin ws 3.1, rnge 1.8to 8.6.In three pre-deliverymniotic fluid smples, density rtiosof lessthn 1.5were found. Those infnts developed RDS. Additionl Keyph rses thin-lyer chromtogrphy densiiometry respirtory distress syndrome #{149} pulmonry surf c/nt Phosphtidyl choline (lecithin) hs been identified s mjor lipid of the surfctnt, lipoprotein whose surfce ctivity imprts stbility to the norml pulmonry lveolus (1). Surfce properties of both lung nd lveolr phosphtidyl choline hve been shown to chnge during mturtion of the humn fetus (, 3). Lung extrcts of infnts dying of respirtory distress syndrome (RDs) disply bnorml surfce ctivity nd contin less phosphtidyl choline (4-6). A mens for the ssessment of the biochemicl mturity of the fetl lung hs evolved from the study of phospholipid content of trchel fluid (7) nd mniotic fluid (8) t vrious fetl ges, nd in norml nd bnor- From the Deprtments of Chemistry, Peditrics, nd Obstetrics & Gynecology, The Mount Sini Hospitl nd The Mount Sini School of Medicine of The City University of New York, New York, N.Y Presented in preliminry form t the 23rd Ntionl Meeting, AACC, Settle, Wsh., August 8-13, Received Mr. 24, 1972; ccepted My 14, ml pregnncies. Although the sphingomyelin concentrtion of mniotic fluid chnges reltively little throughout pregnncy, the mount of totl phospholipid increses s gesttion progresses. This is ttributed to sudden surge of phosphtidyl choline, minly diplmitoyl lecithin, production t bout the 35th week of gesttion. The density rtio of phosphtidyl choline to sphingomyelin in mniotic fluid hs, therefore, been used s mens for ssessment of fetl pulmonry mturity (9). The synthesis of phosphtidyl choline in mmmlin lung occurs vi t lest two mjor pthwys. During erly fetl life tenuous pthwy involving trimethyltion of ethnolmine by S-denosylmethionine ppers ctive (, 10), while in lter fetl life the pthwy involving direct trnsfer of cytidine diphosphte choline to the n-,3-dig1yceride is opertive (3, 11). The determintion of the phosphtidyl choline: sphingomyelin rtio in mniotic fluid my be useful in differentiting the fetus with biochemiclly mture lung from the fetus with biochemiclly immture lung. The ltter is t significnt risk of developing RDS becuse of dependence on the mrginl tnmethyltion pthwy. We developed convenient modified method for the routine determintion of mniotic fluid phosphtidyl choline: sphingomyelin rtio, nd the clinicl significnce of the vlues obtined re evluted. Mterils nd Methods Regents All solvents were of Spectrnlyed grde nd regents were of nlyticl grde. The sphingomyelin stndrd ws obtined from Serdry Reserch Lbortories, P.O. Box 5036, London, Ontrio, Cnd. All other phospholipid stndrds were from Applied Science Lbortories, Inc., Stte College, P Phospholipid stndrd mixture A consisted of 2.5 mg of phosphtidyl choline nd 1.25 mg of sphingomyelin per 1 ml of chloroform: methnol (19:1 by vol); stndrd mixture B contined 1.25 mg of phosphtidyl serine, 1.25 mg of sphingomyelin, 2.5 mg of phosphtidyl choline 956 CLINICAL CHEMISTRY, Vol. 18, No. 9, 1972

2 nd 1.25 mg of phosphtidyl ethnolmine per milliliter of chloroform: methnol. For thin-lyer chromtogrphy, solvent system A consisted of chloroform-methnol-30% NH4- OH-wter (34:14:2:0.75 by vol); solvent system B ws similr mixture without wter (12). The solvent systems were freshly prepred every dy. The detecting regent ws modified Ziminski regent (13): 100 ml of n queous solution of mmonium sulfte (300 g/liter) cidified with 12 ml of concentrted sulfuric cid. Supplies nd Equipment Precoted pltes: Silic-Gel F-254 ( Silpite F-52, 5 X 20 cm, 0.25-mm lyer thickness; Bninkmn Instruments, Inc., Westbury, N.Y ). Trnsmission densitometer: Model R-1 10 (Beckmn Instruments, Fullerton, Clif ). Procedures Extrction. Amniotic fluid smples were centrifuged t 10,000 rpm (12,000 X g) for 10 mm nd the superntnt fluid ws seprted. For lipid extrction 2 ml of the superntnt fluid ws mixed with 2 ml of dry methnol nd 4 ml of chloroform in stoppered tube, nd shken on vortex-type mixer for 1 mm. The milky emulsion ws seprted by centnifugtion. The queous (upper) lyer ws discrded nd the chloroform lyer ws dried with bout 150 mg of nhydrous mgnesium sulfte for 10 mm. It ws then filtered through cotton in 25-mm microfurinel into 30-mi per-shped flsk. The residul mgnesium sulfte ws wshed twice with chloroform nd the pooled solution ws evported in rotry evportor t 50#{176}C. The residue ws dissolved in 90 il of chloroform: methnol (19:1). Thin-lyer chromtogrphy nd densitometry. The precoted pltes were stored in glss cbinet over sturted queous clcium chloride t 32% reltive humidity. About 60 nd 30 d of the sme extrct were pplied, with 2-tel cpillry pipets, to the centers of the strting lines on two pltes, 2 cm from their lower edges. Two microliters of stndrd mixture A ws pplied on one side of ech extrct, 2 1 of stndrd mixture B on the other. Two tnks were linedwith filterpper. One tnk contined 50 ml of solvent A, the other 50 ml of solvent B. The pltes were developed in solvent A until the solvent front rose 10 cm from the points of ppliction. After the pltes were dried t room temperture for 5 mm, they were plced in solvent B nd developed until the solvent front rose 8 cm from the points of ppliction. After drying in ir t room temperture, the pltes were immersed for 1-2 s in horiontl position in glss tnk contining the modified Ziminski regent. The excess liquid ws wiped off the bck of the pltes nd they were plced in preheted oven t 213 ± 2#{176}C for 10 mm. The pltes were cut to 10-cm length nd plced in plstic protective envelopes. The reltive densities of the phosphtidyl choline nd sphingomyelin were quntitted on the densitometer with 1.0 )< 0.4 mm slit nd wvelength of 600 nm. The bseline ws djusted to 100% trnsmittnce on the lightest one between the phosphtidyl choline nd sphingomyelin spots. The densities were recorded s integrtor trce units. ResuIts Methodology Stndrd curves indicted tht the densitometric responses were liner up to 15 ig of phosphtidyl choline nd 4 jtg of sphingomyelin. When lipid concentrtions were plotted vs. their densities, the slopes were different nd vried from plte to plte. However, when incresing rtios of the phosphtidyl choline: sphingomyelin concentrtions were plotted ginst the rtios of their densities, the curve ws liner nd the slope remined constnt (Figure 1). Amniotic fluid lipids were extrcted with single nd multiple extrctions from the sme smple. Although the totl mount of lipids obtined with multiple extrctions ws greter, the phosphtidyl choline: sphingomyelin rtio remined the sme for both the single nd multiple extrctions. In replicte extrctions the coefficient of vrition of the phosphtidyl choline: sphingomyelin rtios ws 8.4%. We nlyed seril dilutions of n mniotic fluid smple obtined from n erly pregnncy with low rtio nd nother from ner term pregnncy with high rtio. In both cses the phosphtidyl choline nd sphingomyelin densitometric responses decresed linerly with the dilutions, while their rtios remined the sme (Figure 2). Two typicl chromtogrms nd their densitometric quntittions re shown in Figure 3. In superntnt fluids stored t -20#{176}Cfor 2-4 dys, the rtios remined the sme. Prllel determintions with nd without cetone precipittion showed no significnt difference (P > 0.4) in the criticl rnge of rtios between 1 nd 2 (9) (Tble 1). CliniclCorreltion Two hundred nd twenty one smples obtined t vrious gesttionl ges were nlyed. Before the 30th week, the rtios rnged from 0.3 to 1.5, between the 30th nd 35th week from 0.3 to 3.6, nd fter 35 weeks from 0.7 to 8.6. In our grouped dt, stndrd sttisticl procedures could not be CLINICAL CHEMISTRY, Vol. 18. No. 9,

3 SI. (A) RATIOS I. (A). 2II 3.. hi 3. 2#{149}I $ i8 MICROGRAMS 3.. l. I ml () 4. RATIOS () SN 0 4 2I. $ hi 2 4 S $ Ii WEIGHT RATIOS OF PHOSPHATIbYL CHOLINI: SPINGOMYRLIN STAICAROS Fig.1.A, Stndrdcurvesfordensitometryof phosphtidyl choline (pc) nd sphingomyelin(s).ech pointis the vergeof 3 determintions. B, Incresingweight rtios of pc nd s stndrds re liner with the rtios of their densities SI ml Fig.2. Seril dilutions of n mnioticfluidwith low densityrtio(a) nd mnioticfluidwith highdensity rtio(b) Aliquots of 0.5, 1.0, 1.5, nd 2 ml were extrcted. The density rtios remined the sme in these concentrtions used becuse of the positive skewness of the frequency distributions. Therefore, for ech group we present the geometric men nd the rnge of distribution (Tble 2). Seril determintions in 12 pregnncies showed tht the rtio my increse s erly s the 32nd week of gesttion nd s lte s the 39th week (Figure 4). The rtios of phosphtidyl choline to sphingomyelin in the mniotic fluid of ll infnts who did not develop lids (regrdless of gesttionl ge) were greter thn 1.8, with positively skewed frequency distribution, while the rtios of the infnts who developed RDS were ll less thn 1.5 (Tble 3). Discussion Gluck et l. (9) ssessed pulmonry mturity by exploiting chnges in phospholipids in mniotic fluid during fetl development. Our modifiction of their procedure is designed to increse the ese nd efficiency of the nlysis. We shortened the procedure by omitting the cetone precipittion step. Chnges in the totl phosphtidyl choline frction were sensitive indictors of pulmonry mturity, s reported by others (14, 15). We used commercilly vilble precoted thin-lyer pltes. Attempts to eliminte binders were unsuccessful becuse of problems in hndling. We did not use the clcium sulfte binder becuse of its lod effect (16). The orgnic binder incorported in the pltes used in our lbortory limited chrring temperture, becuse elevted tempertures resulted in yellow bckground tht interfered with densitometry. In greement with previous reports (17, 18), lowering the chrring temperture pro- Tble 1. Pired Determintions of Phosphtidyl Choline:Sphingomyelin Rtios with nd without Acetone Precipittion Density Smple no No cetone precipittion Acetone precipittion CLINICAL CHEMISTRY, Vol. 18, No. 9, 1972

4 Tble 2. Phosphtidyl Choline:Sphingomyelin Rtios during Gesttion Weeks of gesttion <30 No. smples Group I Group II Group Ill >35 Rtio Infnts without RDS (n = 117) Geometric men, Rnge, Infnt8 with RDS (n = 3) Ptient BA J.T M. F SevereRDS, infnt died. I Mild to moderte RDS, infnt recovered. 29 Phosphtidyl choline:sphingomyelin rtios Geometric men Rnge Tble 3. Predelivery Phosphtidyl Choline: Sphingomyelin Rtios of 120 Pregnncies C I Fig.3.Chromtogrms of mniotic fluidphospholipids indicting(a) biochemicllyimmture nd (B) mture lung Extrcts were pplied in the center rows, stndrd mixtures of phosphtidyl serine (ps), sphingomyelin (s), phosphtidyl choline (pc), nd phosphtidyl ethnolmine (PE) on one side, snd c on the other side A duced liner clibrtion curves without serious bckground interference. Appliction of stndrd mixtures on ech plte controlled reproducibility of the density rtios. In spite of some edge effect (19) the identifiction of the frctions ws cler. Chromtogrphing two quntities of extrcts simultneously ssured tht on the first run, phospholipid concentrtions were on the liner portions of the stndrd curve. Dipping the pltes in the modified Ziminski regent eliminted the 50% sulfuric cid spry nd obvited the need for n exhust system. While other nlyticl vlues for mniotic fluid-such s cretinine content-pper relted to gesttionl ge, the phosphtidyl choline: sphingomyelin rtio ppers to relte to biochemicl mturity of the fetl lung (9). All newborns upon whom mniotic fluid nlyses were performed were exmined fter delivery for gesttionl ge by physicl nd neurologic en- 2$ 31 II WEllS Fig. 4. Seril nd selected single determintions of mniotic fluid phosphtidyl choline:sphingomyelin density rtiosccordingto gesttionlge X, infnts without TEDs; 0, infnt with TEDSwho survived; nd#{149}, infnts with TEDs who died. See text for comment on cses A, B, nd C teri s well s by mternl history. Although the phosphtidyl choline: sphingomyelin rtio increses during gesttion, the point t which the rtio exceeded 2 (the so-clled surge indicting pulmonry mturity) my occur ny time fter the 30th to 32nd week. An infnt with ll of the chrcteristics of 32-week gesttion (Figure 4A) CLINICAL CHEMISTRY, Vol. 18, No. 9,

5 demonstrted rtio of 2.2., nd did not develop RDS, while n infnt with the gesttionl chrcteristics of 37-week pregnncy hd rtio of 0.7 nd developed severe lids (Figure 4B). It would seem tht the ppernce of pulmonry mturity cn be brupt, s in the cse of ptient C (Figure 4), where the rtio rose from 1.1 to 3.6 in three dys. In the 117 infnts who did not develop RDS, ll rtios were greter thn 1.8. In the three infnts in this series who developed lids, ll of whom hd to be delivered by cesren section, the rtios were 0.7, 1.2, nd 1.4. It ppers tht there is little dnger of lids in n infnt delivered fter obtining n mniotic fluid phosphtidyl choline: sphingomyelin rtio greter thn 2. We hve insufficient dt to permit definite sttement regrding those born with rtio of less thn 2. In this ltter group re the neontes with the gretest risk of developing RDS. Although ll infnts with rtios of less thn 2 my not inevitbly develop lids, the decision s to the optiml time to perform delivery in this group should be mde by blncing this risk ginst the other clinicl considertions. We cknowledge the help of Zoltn Sry in obtining mniotic fluid smples nd providing prentl informtion while he ws ffilited with our institution. We re lso grteful to Kir Sell, Ev Gl, Mri Schy, nd Joseph A. Kovcs for their skillful technique in performing the determintions. References 1. Klus, M. H., Clements, J. A., nd Hve!, R. J., Composition of surfce-ctive mteril isolted from beef lung. Proc. No4. Acd. Sci. U.S. 47, 1858 (1961). 2. Gluck, L., Motoym,E. K., Srnits, H. L., nd Kulovich, M. V., The biochemicl development of surfce ctivity in mmmlin lung. I. Pedil. Rc. 1, 237 (1967). 3. Gluck, L., Scribney, M., nd Kulovich, M. V., The biochemicl development of surfce ctivity in mmmlin lung. II. Pedi& Rc. 1, 247 (1967). 4. Gruenwld, P., Johnson, H. P., Husted, R. F., nd Clements, J. A., Correltion of mechnicl properties of infnt lungs with surfce ctivity of extrcts. Proc. Soc. Ep. Bil. Med. 109, 369 (1962). 5. Pttle, R. E., Clireux, A. E., Dvies, P. A., nd Cmeron, A. H., Inbility to form lung lining film s cuse of respirtory distress syndrome in the newborn. Lncet ii, 469 (1962). 6. Avery, M. E., nd Med, J., Surfce properties in reltion to telectsis nd hyline membrne disese. Amer. J. Dis. Child. 97, 517 (1959). 7. Adms, F. H., nd Fujiwr, T., Surfctnt in fetl lmb trchel fluid. J. Pedit. 63, 537 (1963). 8. Nelson, G. H., Amniotic fluid phospholipid ptterns in norml nd bnorml pregnncies. Amer. J. Obtet. Gynecol. 105, 1072 (1969). 9. Gluck, L., Kulovich, M. V., Borer, R. C., Brenner, P. H., Anderson, G. G., nd Spellcy, W. N., Dignosis of respirtory distress syndrome by mniocentesis. Amer..1. Obstet. Gynecol. 109, 440 (1971). 10. Bremer, J., nd Greenberg, D. M., Methyltrnsferring enyme system of microsomes in the biosynthesis of lecithin (phosphtidyl choline). Biochim. Biophy. Act 46, 205 (1961). 11. Kennedy, E. P., nd Weiss, S. B., The function of cytidine coenymes in the biosynthesis of phospholipids. I. Blot. Chem. 222, 193 (1956). 12. Cuner, M. L., nd Dvison, A. N., Quntittive thin lyer chromtogrphy of lipids. J. Chromtogr. 27, 388 (1967). 13. Ziminski, T., nd Borowski, E., A new spry regent replcing sulphuric cid in thin lyer chromtogrphy..1. Chr&-,ntogr. 23, 480 (1966). 14. Adms, F. H., Fujiwr, T., Emmnouilides, G. C., nd Rih, N., Lung phospholipids of humn fetuses nd infnts with nd without hyline membrne disese. J. Pedit. 77, 833 (1970). 15. Bhgwnni, S. G., Fhmy, D., nd Turnbull, A. C., Prediction of neontl respirtory distress by estimtion of mniotic fluid lecithin. Lncet 1, 159 (1972). 16. Skipski, V. P., Peterson, R. F., Snders, J., nd Brcly, M., Thin lyer chromtogrphy of phospholipids using silic gel without clcium sulfte binder. J. Lipid Rc. 4, 227 (1963). 17. Nutter, L. J., nd Privett, 0. 5., An improved method for the quntittive nlysis of lipid clsses vi thin lyer chromtogrphy employing chrring nd densitometry. J. Chromtogr. 35, 519 (1968). 18. Adms, G. M., nd Sllee, T. L., A rpid method for reltive quntittion of lipid clsses seprted by thin lyer chromtogrphy. J. Chromtogr. 54, 136 (1971). 19. Sthl, E., Dunnschicht-Chromtogrphie. IV., Arch. Phrm. (Weinheim) 292, 411 (1959). 960 CLINICAL CHEMISTRY, Vol. 18, No. 9, 1972