Phosphatidylinositol and phosphatidylglycerol in amniotic fluid: Indices of lung maturity

OBSTETRICS

Phosphatidylinositol and phosphatidylglycerol in amniotic fluid: Indices of lung maturity MIKKO

HALLMAN,

MARIE

KULOVICH

ELSA

KIRKPATRICK

ROBERT

G.

LOUIS La Jo/la

M.D.

SUGARMAN,

GLUCK,

M.D.*

M.D.

and Los Angeles,

Calijornia

The minor phospholipids in amniotic fluid from normal pregnancies were correlated with the rcv&establi.~hed index of lung maturity, the lecithinlsphingomyelin (L/S) ratio. When &a LIS ratio was less than 1.0, the acidic phospholipids phosp~tidvlg~cerol (PG) and phosphatidyli,lnsitol (PI) were absent or low in concentration (0.0 to 2.5 per cent of lipid phosphorus). Parallel to the increase in the LIS ratio to 2.0, the content of PI increased to 6.0 to 8.5 per cent of lipid phosphorus. PG, the unique phospholipid of lung surfnctant, first appeared and PI concomitantly decreased when the L/S ratio exceeded 2.0, indicczting the secretion of mature lung surfactant. Analysis of PI and PG in amniotic @id as murkers of surfactant seems to be of value as an additional index of prenatal rwaluation of lung maturity and rna~~be pnrticularlg useful when the specimen is contaminated

with

blood.

From the Department of Pediatrics, UniuersiQ of Cal$ornia, San Diego, and the Division of Maternal-Fetal Medicine, Department of ObstetricsiGynecology, Los Angeles County-Universig Southern California Medical Center. Supported principally by funds from National Health Grants HDO4380, SCOR HLI4169, TW1)2051.

THE LEC:ITHIN/SPHINGOMYELIN (L/S) ratio in amniotic fluid is a widely accepted test for pulmonary maturity of the fetus to prevent the respiratory distress syndrome (RDS) of the newborn infant.rs5The bases of the L/S ratio are: first, that there is deficient surfactant in RDS”, 7; second, that secretion of fetal lung fluid containing surfactant contributes to amniotic fluid’* ‘; third, that the biosynthesis of surfactant lecithin matures at about 35 weeks’ gestation in the normally developing fetal lung.7 The surfactant complex lines alveoli and reduces surface tension on expiration. Thus, the acini are stabilized. Nearly 85 per cent of surfactant is phospholipid which, unlike other body phospholipids, contains highly saturated fatty acids.“’ Lecithin contributes nearly 80 per cent of the surfactant phospholipids. Other minor components

of

Institutes and

of

‘4 preliminary report was presented at the Eighth Annual Meeting of the Society for the Study of Reproduction, Ft. Collins, Colorado, July, 1975. Rereirwd

for

publication

Accepted

Februa~

October

21, 1975.

27, 1976.

Reprint requests: Dr. Louis ScImol of MedirinelC-019, D&go, La Jolla, California

Gluck, Professor of Pediatrics, University of California, San 92093.

*Present address: Department of ObstetricslGynecology, Uniz>ersity Hospital, Saskatoon, Saskatchewaan, Canada.

613

614

Haltman

et al.

phosphatitlylinositol (PI), phosphatitl~lsphingomyrliil. antI phospharitl~lscrinc. ‘l‘hc sccor~l major phospholipid is phosphaidylglycerol (PC;), comprising more than IO pc’r cc111 of. sudxtant phospholipids in thr adult.“~ I2 Ailim;il strurtul-es other than surface-tant c,ontain onlv t raccy of’ this phospholipid. Studies on the pcrinatal development ot Iutig \urfA( tant in the rabbit revealed that in t hr prctcarm Lttus PC; was absent and PI was unusually prominvn~. ‘I‘lleI-cilti~r~. I’(; appear-cd and 1’1 conc.omitantl~ tlcc~rcasetl. ‘“‘I‘hc present report is a study showing similatdevelopment in the human subject and COrrehteS PI and PC; with the functional maturit\, of the lu11,q. ill
~~tt~;il~OlillI~iI~~.

Material and methods Sixty-six amniotic fluid specimens from 44 women were obtained by transabdominal amniocentesis strictI> for maternal and/or fetal indications. Cases of maternal diabetes tnellitus and hypertensive diseases were not included. None of the mothers received corticosteroid derivatives. Each amniotic fluid specimen of .i ml. or more leas centrifuged at 800 x g for five minutes. The lipid extract derived from the supernatant was divided into two parts. In one part, one L/S ratio and the per cent of total lecithin that precipitated in colt1 acetone were measured by reflectance densitomet.ry OII thin-layelplates of silica gel H cctntaining .i l)er cent (NH,),SO, as described by Cluck and associates.‘1 The other part was analyzed for phospholipids with plain silica gel H plates. To increase the resolution of individual comptr nt’nts, thin-layer chromatograms were developed twice. each time in a different dimension. The solvents used in two-dimensional chromatography were as follows: first solvent system, (1) tetrahydrofuran-methylalmethanol-2N NH,OH (10-3-2-1.1, v/v) and (2) chloroform-rnethanol-H,O (65-25-4. v/v); second sol-

vent system, ( I) thlorcJform-meth;Itlol-jH per c cnt NH&H-H20 (130-60-5-4, vi\-) and (2) chtoroforrnmethanol-acetic acid-Ho0 (160-50-I 2-3, v/v). A simplified solvent system developed by R. Bustos and M. V. Kulovich occasionally was used. Between the individual runs, the plates were heated at 70” C. for five minutes. After the development of the chromatograms and the visualization with iodine, the phospholipid phosphorus was measured by a modification’” of the Bartlett method.

Results The molar contents of individual phospholipids were expressed as percentages of total phospholipid. ‘The actual concentrations of individual phospholipids in amniotic fluid also were calculated, but the scatter of’ the results was considerable (data not shown). This may be due to individual variation in amniotic fluid volume, a factor that is obviated when the results are expressed on the basis of‘ total phospholipid. The phospholipids analyzed Mere PI, PG, phosphatidylethanolamine, phosphatidylserine, lecithin and sphingomyelin. In addition. some other phospholipids could be identified. However, their content totaled 3.5 per cent or less of total phospholipid phosphorus. In 20 of t-l cases. samples wert’ obtained within i:! hours prior to birth. Of these, I5 had an L/S ratio of 2 or tnore and no RDS when barn. Of‘ the five with I./S ratios less than 2.0, four had RDS. The parameters measured were correlated to either L/S ratio or 1”’ cent of acetone precipitable over total lecithin. The material was divided into three categories on the basis of PI and PG content (Table I). In the first, PI concentration was less than 4 per cent of total phospholipid and PC; was practically absent. In this category. L/S ratios were low, exceeding 1 in only three of 24 cases. In the second, more than 4 per cent PI was

Volume Number

PI and PG in amniotic fluid

125 5

present and PC was less than 0.5 per cent. Here, L/S ratios were transitional or mature (lowest value, 1.0; highest, 3.1). In the third category, the specimens had both PG and PI. Here, L/S ratios always were mature. The changes in concentrations of phosphatidylethanolamine and phosphatidylserine were less marked except in the third group where their contents were significantly less than in the two other groups. Fig. 1 shows concentrations of PI as a function of the L/S ratio. The contents increased with an increasing L/S ratio up to 3. Thereafter? PI levels gradually fell. Fourth-degree regression analysis showed a significant correlation with an r value of 0.860. At an L/S ratio of 2, the most likely PI content was 7.2 per cent of lipid phosphorus. The relationship between PG and the L/S ratio is illustrated in Fig. 2. With an L/S ratio less than 2, PC; was practic.ally absent. As the L/S ratio increased, PG appearecl. Linear regression analysis showed a significant correlation with an r value of 0.873. The sum of PI and PG correlated with the L/S ratio (data not shown). ‘The sum increased sharply until the L/S ratio reached the value of 3. Thereafter, the increase was small despite increasing amounts of PG due to the simultaneous decrease in PI. Third-degree regression analysis revealed a remarkably good correlation between the L/S ratio and these acidic phospholipids with an r value of 0.953. Contents of PI and PG in amniotic fluid during normal pregnancy are shown in Fig. 3. After 30 weeks, PI increases, peaks at 36 to 37 weeks, then gradually decreases. PG appears after 35 weeks’ gestation and continues to increase beyond term. Fig. 4 describes the L./S ratio, the PGlPI ratio, and the sum of PI and PG as a function of gestational age. The sum of these acidic phospholipids has been expressed on the basis of phosphatidylethanolamine rather than total phospholipid. Phosphatidylserine and sphingomyelin also could be used as reference compounds, similar to phosphatidylethanolamine (data not shown). The sum of PI and PG (divided by phosphatidylethanolamlne) increases parallel with the L/S ratio, exceeding 1 at an L/S ratio of 2. The PGiPI ratio starts to increase after 35 weeks of gestation. At term, it exceeds 1 and three weeks later approaches or exceeds 3.

Comment The present study demonstrates the stepwise appearance of PI and PG in amniotic fluid during the last trimester of normal pregnancy. Studies on lung effluent of newborn babies15and the isolated surfactant complex from perinatal rabbitsi indicate an analogous

615

14

LECITHIN/SPHINGOMYELIN

RATIO

1. Relationship between densitometrically measured L/S ratios from acetone-precipitable fractions and PI content as determined by phosphorus (Z’) analyses. Fourth-degree regression line is shown.

Fig.

0

2

4

6

8

IO

LECITHIN/SPHINGOMYELIN

I2

14

16

I8

RATIO

Fig. 2. Relationship between densitomerrically determined L/S ratios from acetone-precipitable fractions and PG content assayed by phosphorus (P) measurements. Linear regression line is shown (y = 0.71 x -0.63).

stepwise development of these same acidic phospholipids. This and the virtual identity of the unique phospholipid compositions between term amniotic fluid and purified lung surfactant further confirm that the phospholipids in amniotic fluid originate mostly from the fetal lung. Would the measurement of PI and PG in amniotic fluid be valuable as additional indices of lung maturity? PG appears when the L/S ratio exceeds 2 (Fig. 2). Substantial quantities of PG indeed indicate lung maturity. However, its absence does not necessarily mean that RDS is inevitable. We have documented cases where PG first appeared within two hours after birth with no symptoms of RDS but with mature L/S ratios. On the other hand, surfactant with prominent PI sometimes was recovered from lung effluent at birth, but PG did not appear and RDS developed. In these cases the L/S ratio was less than 2 or factors

616

Hallman

et al.

July

Am. J. Obstet.

0 20

25

30

WEEKS Fig. 3. The content phospholipids were percentages of total for each point.

35

40

GESTATION

of PI (0) and PG (o) iu amniotic Huid during normal gestation. The quantified by measuring the phosphorus (P) content and expressed as lipid phosphorus. Means i standard deviations of 3 to 5 samples are shown

p-x

I

1

20

1

I

4

I

I

25

z

I

30

WEEKS

35

40

GESTATION

Fig. 4. L/S ratios (X). PC/PI ratios (0). and PI + PG/phosphatidylethanolamine ratios (a) as a function of the gestational age. L/S ratios have been calculated from the acetone-precipitable fraction with reflectance densitometry, and the others were calculated from the total lipid fraction by measuring the phosphorus contents.

1. 19776

Gynecol.

Volume Number

125 5

PI and PG in amniotic

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amniotic to use

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evaluation of lung maturity. the help of Dr. Allen Lein

REFERENCES 1. Gluck,

8.

2.

9.

3.

4.

5.

6.

7.

L., Kulovich, M. V., Borer, R. C., Jr., Brenner, P. H., Anderson, G. G., and Spellacy, W. N.: Diagnosis of the respiratory distress syndrome by amniocentesis, AM. J. OBSTFT. GYNECOL. 109: 440, 1971. Gluck, L., and Kulovich, M. V.: Lecithinisphingomyelin ratios in amniotic fluid in normal and abnormal pregnancy, AM. J. ORSTET. GYNECOL. 115: 539, 1973. Cluck. L... Kulovich, M. V., Borer, R. C., Jr., and Keidel, W. N.: The interpretation and significance of tht lecithinisphingomyelin ratio in amniotic fluid, AM. J, OBSTET. GYNECOL. 120: 142, 1974. Donald, I. R., Freeman, R. K., Goebelsmann, U., Chan, W. H., and Nakamura, R. M.: Clinical experience with the amniotic fluid lecithinlsphingomyelin ratio. I. Antenatal prediction of pulmonary maturity, AM. J. OBSTET. GYNECOI.. 115: 547, 1973. Whitfiel(l, C. R.: Measurement of pulmonary surfactant in amniotic Huid in the assessment of fetal lung development and the risk of neonatal respiratory distress, Eur. J. Obstet. Gynecol. Reprod. Biol. 3: 215, 1973. Avery, RI. E., and Mead, J.: Surface properties in relation to atelcctasis and hyaline membrane disease, Am. J. Dis. Child. 97: 517-523, 1959. Gluck, L.. Kulovich, M. V., Eidelman, A. I., Cordero, L., and Khazin, A. F.: Biochemical development of surface activity in mammalian lung. IV. Pulmonary lecithin synthesis in the human fetus and newborn and etiology of the respiratory distress syndrome, Pediatr. Res. 6: 81, 1972.

10. 11. 12.

13.

14.

15.

16.

Clements, J. A.: Composition and properties of pulmonary surfactant, in Villee, C. A., Villee, D. B., and Zuckerman, J., editors: Respiratory Distress Syndrome, New York, 1973. Academic Press, Inc., p. 205. Cluck, L., Chez, R. A., Kulovich, M. V., Hutchinson, D. L., and Niemann, W. H.: Comparison of phospholipid indicators of fetal lung maturity in the amniotic fluid of the monkey (Macaca mulatta) and’ baboon (Pa,& papia}, AM. J. OBSTET. GYNECOL. 120: 524, 1974. Farrel, P. M., and Avery, M. E.: Hyaline membrane disease, Am. Rev. Resp. Dis. 111: 657, 1975. PHeger, R. C., and Thomas, H. G.: Beagle dog pulmonary surfactant lipids. Arch. Intern. Med. 127: 863, 1971. Hallman, M., and Cluck. L.: Phosphatidyl glycerol in lung surfactant: I. Synthesis in rat lung microsomes, Biochem. Biophys. Res. Commun. 60: 1. 1974. Hallman, M., and Cluck, L.: The biosynthesis of phosphatidylglycerol in the lung of the developing rabbit, Fed. Prod. 34: 274, 1975. Gluck, L., Kulovich, M. V., and Borer, R. C., Jr.: Estimates of fetal lung maturity, Clin. Perinatol. 1: 125, 1974. Hallman, M., Feldman, B., and Gluck, L.: RDS: The absence of phosphatidylglycerol in surfactant. Pediatr. Res. 9: 396, 1975. Cluck, L., and Kulovich, M. V.: The evaluation of functional maturity in the human fetus, in Cluck, L., editor: Modern Perinatal Medicine, Chicago, 1974, Year Book Medical Publishers, Inc., pp. 195-207.