sphingomyelin ratio of amniotic fluid

133

Clinica

Chimica

0 Elsevier

Acta, 64 (1975) 133-142 Scientific Publishing Company,

Amsterdam

- Printed

in The Netherlands

CCA 7273

A STUDY OF THE LECITHIN/SPHINGOMYELIN FLUID

RATIO OF AMNIOTIC

D.O.E. GEBHARDTa’*, J.M.W.M. MERKUSd

F.N. WILDEBOERC

a Department b Laboratory ‘Physiological d Department (Received

A. BEINTEMAa,

R.E. DE ROOIJb,

and

of Obstetrics and Gynaecology, University Hospital, Leiden, of Medical Chemistry, University of Leiden, Leiden, Laboratory, Medical Faculty, University of Nijmegen, Nijmegen and of Obstetrics and Gynaecology, Maria Hospital, Tilburg (The Netherlands)

April 28, 1975)

Summary 1. There is a significant correlation between lecithin/sphingomyelin (L/S) ratios based on densitometry (L/S), and L/S ratios based on phosphorus determinations ((L/S),). 2. The fetal lung is mature when the (L/S), , determined according to Verhoeven, A.G.J. and Merkus, H.M.W.M. (1974) Clin. Chim. Acta 53, 229-232, is 1.2. This value is equivalent to an (L/S)p of 1.8. 3. The acetone precipitation procedure, introduced by Gluck, L., Kulovich, M.V., Borer, R.C. and Keidel, W.N. (1974) Am. J. Obstet. Gynecol. 120, 142-155, is a necessary step for isolating surface-active lecithin. 4. Standardization of the (L/S), test is feasible and should permit different laboratories to use the same transition point.

Introduction The literature on the relationship between certain phospholipids in amniotic fluid and the occurrence of the respiratory distress syndrome (RDS) has shown an exponential growth [l]. In the original paper of Gluck et al. [2] it was concluded that: “A sudden increase in lecithin concentration after 35 weeks heralds maturity of the pulmonary. alveolar lining. Respiratory distress will not occur should the fetus then be born. Clinical interpretation is made on a thinlayer chromatogram by inspection; a lecithin spot clearly larger than that of * Correspondence to: Dr. D.O.E. Gebhardt. University Hospital, Gymecology, Rtinsburgerweg 10. Leiden, The Netherlands.

Department

of Obstetrics and

134

sphingomyelin marks pulmonary maturity in the fetus.” Some time later Gluck [3] added that: “The lecithin/sphingomyelin (L/S) ratio is easily determined, is independent of amniotic fluid volume and is specific in predicting maturity of fetal lungs. When these compounds are compared densitometrically rather than by actual concentrations, a densitometric ratio of two or more represents maturity of the lung and the infant will not have RDS.” The introduction of a densitometric transition point has, however, been criticized by Gebhardt and Dubbeldam [4]. In a more recent paper Gluck and Kulovich [ 53 come to the surprising conclusion that: “Densitometric ratios are significantly more reliable and far less variable than concentration ratios. At maturity an L/S ratio of 2 : 1 by density comparison actually reflects a ratio of concentrations around 4 : 1.” An important aspect of Gluck’s work 163 which has not found universal acceptance, concerns the question of whether it is possible to separate surfaceactive lecithin from its non-active component by precipitation with cold acetone. In their first paper on the development of surface activity in the fetal rabbit lung, Gluck et al. [7] reported that only acetone-precipitated phospholipids showed low surface tension. Therefore, when Gluck et al. introduced the L/S determination into obstetrics, acetone precipitation was considered indispensable although subsequently some workers have omitted this step [8-lo]. Another problem which has not received the necessary attention concerns the quality of the lecithin and sphingomyelin standards obtained from commercial dealers. These substances serve mostly only for localization of the amniotic fluid phospholipids on the thin-layer chromatograms. For purposes of standardization however, an evaluation of the L/S ratio of the markers is desirable and this is possible only if the purity and structure of the lecithin and sphingomyelin are known. Dipalmitoyl lecithin can be purchased as a synthetic product of high purity. Sphingomyelin, on the other hand, is only available as a chromato~aphically homogeneous extract from bovine brain. The various questions raised in this introduction can be formulated as follows: 1. Is there a significant correlation between the densitometric L/S ratio, content, (L/S),, and does (L/S), , and the L/S ratio based on the phorphorus the coefficient of variation of the (L/S), determination differ from that of the (L/S), determination? 2. Is it true that at maturity the (L/S), is 4 : l? 3. Is it possible to separate the surface-active lecithin from the inactive form by means of acetone precipitation? 4. Do standards from various commercial sources have the same fatty acid composition? Materials and methods The (L/S), determinations were performed as described by Verhoeven and Merkus [ll]. The method consisted of centering the ~niotic fluid (10 min at 2000 X g) and extracting the phospholipids with methanol/chloroform (1 : 2, v/v). The extract was then evaporated to dryness and the residue dissolved in adrop of chloroform. After precipitation with acetone, the phosholipids were separated by thin-layer chromatography (TLC) on precoated glass

135

plates (Merck No. 5714) or on precoated plastic foil (Merck No. 5748). Lecithin and sphingomyelin were visualized with the phosphomolybdic acid spray and their ratio was determined densitometrically in transmission. For the data compiled in Table I, use was made of a Vitatron TLD 100 flying spot densitometer. All other measurements were done with the Chromoscan MK II doublebeam recording densitometer. Since the precoated glass plates did not fit in the Chromoscan, plastic foil TLC sheets, cut to a suitable size, were used with this instrument. The (L/S), determination was based on the method described by Bhagwanani et al. [12] who estimated lecithin in amniotic fluid. Both sphingomyelin and lecithin were precipitated with acetone prior to TLC analysis. The phospholipids on the chromatograms were localized with iodine vapour. The bands were scraped from the plates and the scrapings were transferred directly to the tubes in which the perchloric acid digestion was performed. The isolation of lecithin for the fatty acid analysis and for the measurement of surface activity proceeded according to Kates [13] using 40-week amniotic fluid. Two minor modifications were introduced: (1) the chromatographic absorbent was silicic acid (Merck No. 7754) and not Florisil, (2) the column was eluted with the following solvent mixtures: chloroform/methanol, 95 : 5; 90 : 10; 80 : 20; and 70 : 30. The composition of the eluate was checked by thin-layer chromatography. Lecithin free of other phospholipids was found in the chloroform/methanol 70 : 30 fraction. Analyses of the fatty acid methyl esters of lecithin and sphingomyelin were done by dual-column gas liquid chromatography on a Pye GCV instrument. Transesterification was performed by treating 2 mg of the phospholipid with 3 ml methanol half saturated with gaseous hydrochloric acid. This mixture was refluxed for 3 hours at 80°C and then extracted with 3 ml chloroform and 3 ml water. The chloroform layer was washed with water and then used for gas chromatography. The separation took place on a 1.1 meter glass column packed with 10% SP 2300 on SO/l00 Supelcoport. The column was equipped with a hydrogen flame ionization detector operating at 290°C. The injection temperature was 250°C and the analyses were made by programming from 160°C to 250°C with a 0.7”C/min rise in temperature. Identification of the fatty acid methyl esters was done on the basis of their retention time using authentic substances as reference. For quantitation an Infrotronics automatic electronic integrator model 208 with digital printout was used. The phospholipids needed for studying the quality of controls (dipalmitoyl lecithin, egg yolk lecithin, sphingomyelin) were purchased from respectively Koch-Light, Colnbrook, England and from Sigma, St. Louis, MO. U.S.A. Surface tension was measured with the modified Wilhelmy balance of Schoedel et al. [ 141. 100 ~1 lecithin (1 mg/ml) dissolved in hexane/ethanol 4 :1 were layered over tap water of a temperature varying between 19°C and 24°C. The balance was cycled between a maximum (100% = 100.9 cm:) and a minimum area (20% = 21.7 cm2) until there was no further reduction in the minimum surface tension (10th cycle). For continuous measurements of the surface tension and surface film area, the balance was connected with an XY recorder. The cycle time from the maximum area to the minimum and back to the maximum was 20 seconds.

136

The amniotic fluid analyses shown in Table I were derived from 225 samples obtained at amnioscopy. This was done during the last 24 hours before parturition. The 36 amniotic fluid samples used for establishing the correlation between (L/S), and (L/S)p values, (Fig. l), were obtained by amniocentesis at different stages of pregnancy. Results In Table I the relationship between the (L/S), ratios and the occurrence of RDS is shown. A transition point of 1.2 was introduced since the highest (L/S), associated with RDS in the neonate was 1.1. In Fig. 1 the correlation between (L/S), and (L,k$ ratios and also the regression equation are presented. It is seen that the (L/S), transition point of 1.2 is equiv~ent to an (L/S), of approx. 1.8. The reproducibility of the (L/S), determination, expressed as the coefficient of variation, was 8% when the ratio was 1.2 -(n = 10). For an (L/S), of 1.8 the coefficient of variation was 10% (n = 10). TABLE

I

The relationship between the (L/S)Q ratios of 225 amniotic and the respiratory condition of the neonate.

(L/S)D (L/S)D

4 1.20 3 1.20

fluids, obtained

within 24 hours of parturition,

Anectasis

RDS succumbed

RDS survived

No RDS normal

2 0

13 0

I 0

3 200

r - 0.93 y’ 1.4x + 0.109

Fig. 1. The relationship between densitometric of these phospholipids in amniotic fluid.

L/S ratios and L/S ratios based on the phosphorus

content

137

2 I

A

B

z

Fig. 2. Thin-layer chromatogram of: A. An amniotic fluid extract after acetone precipitation. B. An extract from the same amniotic fluid without acetone precipitation. The chromatographic separation and staining were performed according to Verhoeven and Merkus [ll] . The bands are: 1, Phosphatidyl serine + lysolecithin; 2, Sphingomyelin (double band); 3. Lecithin, the arrow indicates the faster moving moiety; 4. Phosphatidyl ethanolamine.

The influence of acetone treatment was studied in the first place by comparing the chromatographic separation of an acetone-precipitated (Fig. 2A) with an acetone-untreated phospolipid extract (Fig. 2B). The main difference between the two chromatograms resides in the breadth of the lecithin bands. It appears that in the extract which was not treated with aceton, the lecithin con-

Fig. 3. A comparison between the migration speed of: A. Acetone-soluble + acetone-insoluble lecithin isolated from alone. E. Acetone-insoluble lecithin alone. The substances zontal line under the letters and chromatography proceeded

Egg yolk lecithin; B, Dipalmitoyl lecithin; C. an amniotic fluid. D. Acetone-soluble lecithin were applied to the pmcoated Plate 0x1a ho+ according to [ill.

138 TABLE

II

% Fatty

acid composition

C(14 C(16 C(16 C(18 C(18 C(18 C(18 C(20 C(22

: 0) : 0) : 1) : 0)

: 1) : 2) : 3) : 4) : 6)

of lecithin

from term amniotic

fluid and from egg yolk,

Acetone-soluble lecithin from term amniotic fluid

Lecithin from egg yolk (Sigma) batch 83C-8140-l

Acetone-insoluble lecithin from term amniotic fluid

1.8 45.8 14.3 5.0 22.2 4.8

0.2 30.0 2.7 12.7 31.8 16.0 0.4 2.8 2.7

3.5 76.2 4.2 2.8 5.3 1.5 0.2 1.1 1.1

0.3 1.2

tains a fraction which travels faster (see arrow Fig. 2B) than acetone-insoluble lecithin. The difference in migration speed is also shown in Fig. 3 where egg lecithin (A), dipalmitoyl lecithin (B), lecithin isolated from a 40 week amniotic fluid (C), the acetone-soluble fraction of C (D), and the acetone-insoluble fraction of C (E), are shown after chromatography. It is clear that dipalmitoyl lecithin (B) and acetone-insoluble amniotic fluid lecithin (E) have a similar migration rate. This also holds true for egg lecithin (A) and acetone-soluble amniotic fluid lecithin (D). Another indication that acetone-insoluble lecithin is similar to dipalmitoyl lecithin, whereas acetone-soluble lecithin resembles egg lecithin, comes from a fatty analysis of these phospholipids (Table II). The acetone-insoluble lecithin

o I 100

80

60

LO

20 O/Area

Fig. 4. Surface tension-area diagram of 0.1 mg acetone-insoluble tinuous line) and of 0.1 mg dipalmitoyl lecithin (stippled line).

lecithin

from

term amniotic

fluid (con-

139

dyn

60

60

80

100

LO

20 %Area

Fig. 5. Surface tension-area diagram of 0.1 mg acetone-soluble tinuous line) and of 0.1 mg egg yolk lecithin (stippled line).

lecithin

from term

amniotic

fluid (con-

contains mostly saturated fatty acids (82%), including 76% palmitic acid, whilst egg yolk lecithin and acetone-soluble lecithin are rich in unsaturated fatty acids. Further evidence that the acetone-insoluble fraction of amniotic fluidlecithin is the biologically active lung surfactant, follows from the surface tension-area diagrams. In Fig. 4 the continuous curve represents such a diagram from compression and expansion of acetone-insoluble lecithin. The stippled

TABLE

III

% Fatty

acid composition

of commercially

Sigma Batch C(14 C(16 C(17 C(18 C(19 C(20 C(21 C(21 C(22 C(22 C(23 C(23 C(24 C(24 C(25 C(25: C(26 C(26

: 0) : 0)

: 0) : 0)

: 0) : 0) : 0) : 1) : 0) : 1) : 0)

: 1) : 0)

: 1) : 0) 1) : 0) : 1)

0.1 3.1 0.2 46.2 0.3 1.6 0.1 < 0.1 9.3 1.7 2.0 1.3 9.2 19.1 1.0 1.3 0.8 2.1

92 C-0890

available

sphingomyelin Koch-Light 0.1 2.5 0.2 44.5 0.8 2.5 0.3 0.1 8.6 2.0 2.2 1.4 8.9 18.2 1.4 1.9 0.9 3.3

from bovine brain. Batch

58550

140

curve was obtained with dipalmitoyl lecithin. In Fig. 5 the surface tension-area diagram of acetone-soluble lecithin (continuous curve) and egg lecithin (stippled curve) are reproduced. It is shown that the minimum tension reached in Fig. 4 is zero, whereas in Fig. 5 the lowest surface tension is 15 dyne/cm. Finally in Table III an analysis is given of the sphingomyelins purchased from Koch-Light and from Sigma. The phosphorus content of both samples was 3.3%. This is somewhat lower than the value reported by the manufacturers and makes it likely that the standard contained traces of another component. It is clear that the fatty acid composition is almost identical in the two batches. The gas chromatographic analysis of the synthetic lecithin samples from Koch-Light and Sigma revealed that both substances were 99% pure dipalmitoyl lecithin. Discussion The results of the experiments have provided answers to the questions raised in the introduction. In the first place there is a close correlation between (L/S), and (L/S)r values (Fig. 1). Although this may not come as a surprise it should be remembered that Gluck [5] considered the (L/S), ratio to be an unreliable measure of lung maturity. It is not clear why he was unable to obtain a better correlation but perhaps his method of isolating lecithin and sphingomyelin on columns of diethyl-aminoethyl cellulose [ 51 is unsuitable for determining (L/S)* ratios. Furthermore there is little difference between the precision of the (L/S), and the (L/S), determinations. When the results of Table I are taken in combination with Fig. 1, it follows that lung maturity can also be established on the basis of an (L/S), transition point. Using the regression equation: (L/S), = 1.4 (L/S), + 0.109 and substituting 1.2 for the (L/S) n one finds an (L/S), of approx. 1.8. This value is far removed from the concentration ratio of 4 : 1 which according to Gluck et al. [ 61 represents the transition point when lung maturity is reached. In general, determination of (L/S), values does have some advantage viz. the tedious procedure of scraping the lecithin and sphingomyelin bands from the TLC plates is avoided. The (L/S), can, however, be useful as a reference for purposes of standardization. When use is made of (L/S), ratios, standards should always be included in the procedure and also evaluated densitometritally. For instance’Verhoeven and Merkus [ll] have shown that a solution containing 2 mg/ml lecithin and 1 mg/ml sphingomyelin will yield an (L/S),, of approx. 1.3. Since the sphingomyelin from Sigma and from Koch-Light have almost the same fatty acid composition (Table III) their staining capacity with phosphomolybdic acid will be similar (Gebhardt and de Rooij [15] ). Furthermore since 99% pure synthetic dipalmitoyl lecithin is available, standardization of the (L/S), technique should no longer be a problem. The great diagnostic value of the (L/S), ratio (Table I) has also been stressed by Gebhardt and Beintema [16] who showed that (L/S), determinations were more useful for establishing lung maturity than measurements of the total lecithin content, or the total lecithin/total phospholipid ratio. (In their paper Gebhardt and Beintema used a transition point of 2.0 because the phos-

141

pholipids were sprayed with 10% sulphuric acid instead of phosphomolybdic acid.) The importance of the acetone-precipitation step becomes evident when Figs 2, 3, 4 and 5 are studied together with the results of the fatty acid analysis (Table II). It is apparent that even with a simple one-dimensional chromatographic separation, two types of lecithin can be distinguished. Firstly a fast fraction with various properties similar to egg lecithin (e.g. the same surface activity and migration rate and a similar percentage of unsaturated fatty acids), and secondly a slower fraction which resembles dipalmitoyl lecithin. It is well known that dipalmitoyl lecithin, contrary to egg yolk lecithin, is insoluble in acetone [17]. Therefore it is difficult to explain the finding of Roux et al. [18] that acetone-soluble and acetone-insoluble lecithin from amniotic fluid have the same fatty acid composition. The possibility exists that by treating the lecithin with dichlorofluorescein before gas chromatography, the C(16 : 1) was transformed to C(16 : 0). This seems all the more plausible since Roux mentions that: “The presence of unsaturated Cl6 is almost negligible at all stages of development”, whereas Ekelund et al. [ 191 and Arvidson et al. [20] have found about 5% C(16 : 1) lecithin in term amniotic fluid. There is undoubtedly great need for standardisation of the Gluck test. Many publications with modifications of the original technique have appeared in which (L/S), values of controls and precision of the method have been omitted. This may explain the reluctance which exists in many laboratories to add the Gluck test to their programme of diagnostic aids. As long as a standardized procedure is not available we agree with Wagstaff et al. [21] that: “The critical values described in published reports should not be adopted without a careful appraisal of the method.” These authors concluded that staining with phosphomolybdic acid followed by transmission densitometry was an accurate and sensitive method for clinical purposes. Our results are in full agreement with such an opinion and we believe that standardization of the (L/S), determination is now feasible, In the future it will be necessary to investigate whether the relatively high coefficient of variation (about 10%) can be reduced. If this is possible, the predictive value of the test, which is already high, will increase even further. Acknowledgements The authors wish to acknowledge the valuable assistance Koen and A.G.J. Verhoeven and also of Miss II. Vreeburg.

of Drs. E.H.

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142 9 Blass. K.G.. Thibert. R.J. and Draisey. T.F. (1973) Clin. Chem. 19, 1394-1396 10 Morrison, J.C., Wiser, W.L., Arnold, SW., Whybrew, W.D.. Morrison, D.L., Fish, S.A. and Bucovti, E.T. (1974) Am. J. Obstet. Gynecol. 120, 1087-1091 11 Verhoeven. A.G.J. and Merkus. H.M.W.M. (1974) Clin. Chim. Acta 53, 229-232 12 Bhagwanani, S.G., Fahmy, D. and Turnbull, A.C. (1972) Lancet ii, 66-67 13 Kates, M. (1972) Techniques of Lipiddogy, p. 401, North-Holland, Amsterdam 14 Schoedel, W., Slama. H. and Hansen, E. (1969) Pfliiger’s Arch. Eur. J. Physiol. 306.20-32 15 Gebhardt, D.O.E. and de Rooij. R.E. (1975) Clin. Chim. Acta 59. 267--268 16 Gebhardt, D.O.E. and Beintema. A. (1975) Am. ,J. Obstet. Gynecol.. 122.734-736 17 Dervichian, D.G. (1964) in Progress in Biophysics and Molecular Biology (Butler, J.A.V. and Huxley, H.E., eds.) Vol. 14, PP. 263-342 18 Roux, J.F., Nakamura, J. and Froslono, M. (1974) Am. J. Obstet. Gynecol. 119, 838-843 19 Ekelund, L.. Arvidson, G. and Asted, B. (1973) J. Obstet. Gynaecol. Brit. Commonw. 80.912-917 20 Arvidson, G., Ekelund, H. and Asted, B. (1972) Acta Obstet. Gynecol. &and. 51, 71-75 21 Wagstaff, T.I., Whyley. G.4. and Freedman, G. (1974) Ann. Clin. Biochem. 11.24-30