Assay of phospholipids in the amniotic fluid

251

Clinica ChimicaActu, 51 (1974) 257-269 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

CCA 6311

ASSAY

OF PHOSPHOLIPIDS

IN THE AMNIOTIC

FLUID

H. VERDER and J. CLAUSEN University Clinics of Pediatrics and Obstetrics, Copenhagen County Hospital, Gentofte, 2900 Hellerup, and Neurochemical Institute, 58 Rbdmandsgade, 2200 Copenhagen (Denmark) (Received October 8,1973)

Summary

A thin-layer chromatographic method for assay of acetone precipitated phospholipids of amniotic fluid of use in prenatal diagnostics is described. The lipids separated on the chromatoplate were quantitatively assayed by a direct densitometric method, and the phospholipids expressed in proportion to sphingom yelin . Blood admixture and activity of lipases in the amniotic fluid will give misleading results. Lipases could be impeded by ethylenediaminetetraacetate. The extraction volume of chloroform-methanol and the evaporation temperature are crucial. Confusion of sphingomyelin and lysophosphatidylcholine could be a source of error.

Introduction

Idiopathic respiratory distress syndrome (IRDS) has a central position in the clinic of the newborn, due to the risk of complications [1,2] . 20-30% succumb, and hyaline membranes are found in 19-30.5% of newborn who die [21* Studies indicate that IRDS is associated with a reduced content of lung surfactants [ 3,4] . Dipalmitoylphosphatidylcholine (DPPC), present in the lungs due to its low surface tension, determines the ability of the newborn’s lung to expand, and the DPPC content in lungs and amniotic fluid (AF) increases during pregnancy [ 51. The purpose of the present work was to develop a quick thin-layer chromatographic (TLC) method for clinical use. The chemical and clinical data are compared in a second paper [6] . Chemicals

stadt,

The chemicals used were of highest obtainable purity from Merck, DarmGermany, with the exception of chromatographically pure phospholipid

258

(P-lipid) markers: lysolecithin, sphingomyelin (from bovine brain), L-a-lecithin (type III-E, from egg yolk) all from Sigma, and phosphatidylethanolamine from British Drug Houses, Poole, Dorset, England. Methods Processing of amniotic fluid AF was centrifuged at 0” at 1100 X g in a “Sorvall superspeed, RC-2, centrifuge” for 5 min, and the supernatant was sucked off, Lipids in the supernatant were either extracted at once, or AF was refrigerated (--20”) for later processing.

1 vol. AF (1-2 ml) was extracted with 5 vol. chloroform-methanol (2:1, v/v) [7] in a whirlmixer (Fisons, England) for 5 min. The mixture was centrifuged at 0” (5 min, 1100 X g), and the bottom-layer transferred to a conical tube using Pasteur pipettes. The chloroform phase was evaprrated to dryness in water bath at 70” in a fast stream of oxygen-free dried nitrogen. Acetone ~reci~i~a~ion The lipid extract was precipi~ted with acetone, in order to separate mainly saturated (surface-active) P-lipid from unsaturated P-lipid [5,8]. Two drops of cold acetone were added to extract from 1-2 ml AF, and a white precipitate was formed after shaking, then a further 750 ~1 cold acetone was added, and after 15 min in an ice bath the tube was centrifuged (3 min, 1100 X g), the supernatant was decanted and the precipitate was dried in a stream of nitrogen.

The acetone precipitate was dissolved in 51tlOO ~1 chloroform (0”) and 25-100 ~1 of the material was applied in a l-cm-broad zone with a Hamilton microsyringe on a glass plate (20 cm X 20 cm) covered with a 0.25-mm-thick layer of Kiselgel G (30 g suspended in 60 ml distilled water) with an adjustable applicator (Desaga Werke, Germany) [ 9 ] , Before use the plates were preheated (1 h, 110”). Chromatograms were developed ascendingly approx. 10 cm (22”) in a chromatotank. Mobile phase: chloroform-methanol-25% aqueous NH, (70:30:5, v/v/v). Detection of lipid spots After drying in open air (22”) the plate was sprayed with ammonium molybdate perchloric acid --HCl reagent [lo], heated at 100” in an oven for 15 mm and then kept for 1 h in the dark (22”) before scanning in a densitometer equipped with an integrating recorder (Vitatron Ltd, Dieren, Holland). A slit width of 0.25 mm, a mercury lamp and a filter of 545 nm were used [ll] . In order to identify the AF P-lipids 10 ~1 of lysophosphatidylcholine (lyso-P-choline), phosphatidylcholine (P-choline), sphingomyelin (Sph), and phosphatidylethanolamine (P-ethanolamine) markers (0.5% w/v) were applied on the plates, and the R, values were compared with those of the sample. In order to further identify the lipids carboxylic esters were identified [12], and

259

two-dimensional TLC was performed on Kiselgel G in a 0.25-mm-thick layer. In the first dimension the chromatogram was developed 8 cm with chloroform-methanol--25% aqueous NH3 (70:30:5, v/v/v), dried for 10 min and developed 8 cm in the second dimension with chloroform-acetone--methanol acetic acid-water (70:20:10:10:5, by vol.). Lipid material (from 2-3 ml AF) was in two-Dimensions TLC applied in a l-cm-broad zone. The spots were identified by their R, values in comparison with those of the markers, and by addition of the individual marker to the AF lipids.

Phosphorus determination To evaluate the linearity of the present TLC method the P-content of the lipids was assayed and compared to the peak area of corresponding samples. P-lipids were initially separated upon Kiselgel H, and c~omat~~ams developed one-dimensionally as described. Lipids were traced with iodine vapour and eluted [13] , and P-content was determined as phosphate [14]. The samples were read in a “Beckman DB Spectrophotometer”. Results In the one-dimensional TLC it was necessary to use lipid markers in order to trace the individual P-lipid fractions. F~thermore it was possible to distinguish between lyso-P-choline and Sph by means of the hydroxamate reaction

Fig. 1. One-dimensional TLC of amniotic fluid P-lipids, (3 X 1 ml of same normal fluid). Mobile phase: Chloroform-methanol-26% aqueous NH3, (70:30:5, v/v/v). The spots were located by (A) ammonium molybdate. (B) iodine vapour, (C) hydroxamate reaction. Abbreviations: or = origin, 1~ = lysophosphatidylcholine (RF = 0.05), s = sphingomyelin (RF = 0.15). 1 = phosphatidylcholine (RF = 0.22). e = phosphatidylethanolamine (RF = 0.33). g = glycolipid (RF = 0.42), fa = free fatty acids, f = front with neutral lipids.

260

I

i, ,/ f I

1

t Ql S OlY 0or

1 0

as c)LY -or

f

Fig. 2. Two-dimensional TLC of amniotic fluid P-lipids from 3 ml normal centrifuged fluid (A) after addition of 10 ~1 sphingomyelin marker (0.5%. w/v) at the orgin, (B) after addition of 10 ~1 lysophosphatidylcholine marker (0.5%, w/v). Abbreviations: see Fig. 1. di = dimethylphosphatidylethanolamine, n = neutral lipids. Mobile phase: first direction (I), chloroform-methanol-25% aqueous NH3 (70:30: 5. v/v/v), second direction (2). chloroformacetone-methanol-acetic acid--water (50:20:10:10:5, by vol.). The spots were located by iodine vapour. Both chromatoplates are in half size.

and two-dimensional TLC (Figs 1 and 2). Lyso-P-choline is present in all normal AF [6] free of blood (Figs 1 and 2). In the following the P-lipids are expressed in proportion to Sph directly as the proportion of the peak areas of the lipids appearing in the one-dimensional TLC after molybdenum spraying. In cases of an insufficient separation of Sph and lyso-P-choline in onedimensional TLC, a wrong L/S* may be obtained. This could be avoided by applying small amounts of lipids (not more than 0.0025 mg/ml of Sph and lyso-P-choline applied in a 0.2 cm X 1 cm zone), and by developing the chromatogram more than 8 cm. Thus with AF from the term, lipid material from 0.5 ml was applied. If the lipid fractions are too weak, as is the case in IRDS or hydramnion, lipid material from 2-5 ml of AF had to be used. Enzymatic and/or chemical deacylation of stored amniotic P-lipids Prior to an evaluation of the clinical validity of assaying P-lipids in AF the optimal storage and treatment conditions have to be elucidated. Thus lipases, if any, in the AF may deacylate the iipids, or this may be effected by an acid eutectic mixture formed during freezing. Furthermore blood admixture and the chemical treatment may influence the results. Table I shows decomposition of P-choline and lyso-P-choline by standing, and also that the decomposition could be inhibited by addition of either trichloroacetic acid (10% w/v) (TCA) or monosodium-ethylenediamintetraacetate (10% w/v, pH 7.4) (EDTA) both of which may inhibit lipases [15] . Deep freezing does not alter L/S (Table II).

*

In the following phosphatidylcholinelsphingomyelin ratio is determined L/S, lysophosphatidylcholinelsphingomyelin ratio LysolS, and phosphatidylethanolaminelsphingomyelin ratio E/S.

261 TABLE I DECOMPOSITION OF P-LIPIDS IN THE AMNIOTIC FLUID The standard procedures of extraction, abbreviations. One sample was assayed.

chromatography

Immediate extraction* Extraction after storage for 24 h at 24O* 0.1 ml 10% (w/v) FnTA added immediately, extraction after 24 h at 24’ * Uncentrifuged amniotic fluid + 0.1 ml 10% [w/v) EDTA immediately** Uncentrifuged amniotic fluid + 0.1 ml 10% (w/v) TCA immediately** Pure amniotic fluid**

and detection of spots are used. See text for

L/S (moles/mole)

LYSOIS

13.2 + 1.1 4.8 f 0.4

4.8 + 0.5 2.3 f 0.2

13.8 + 1.1

4.6 + 0.5

12.8 + 1.0

6.3 f 0.6

7.6 + 0.6 6.1 ?r 0.5

4.8 + 0.5 2.9 + 0.3

(moles/mole)

* 2 ml centrifuged amniotic fluid without blood. ** 5 ml uncentrifuged amniotic fluid without blood, stored at 0’ for 10 days before extraction. TABLE II L/S AND DEEP FREEZING (- 207 See text for abbreviations. LysolS (moles/mole)

L/S (moles/mole)

2.5 ml* with acetone precipitation

Immediate extraction

Deep freezing

Immediate extraction

Deep freezing

4.2 + 0.3

4.6 + 0.4

1.8 + 0.2

2.1 + 0.2

* 2.5 ml centrifuged amniotic fluid without blood. The standard procedure of extraction, chromatography and detection of spots are used.

Influence of blood admixture Blood contamination will alter L/S if L/S is low. Thus more than blood will give quite misleading results (Table III).

1%

Evaluation of optimal conditions for extraction of P-lipids Extraction of AF with chloroform-methanol (2:1, v/v) in the proportion of 1:5 (v/v) shows a maximal extraction of lipids (Table IV). At further reTABLE III L/S AND BLOOD ADMIXTURE

TO THE AMNIOTIC FLUID

Blood admixture in per cent (v/v)

L/s* (moles/mole)

0

1

1.5

2

3

1.1 * 0.1

1.9 f 0.2

2.2 +_0.2

2.4 + 0.2

2.1 + 0.2

* Amniotic fluid from a patient with IRDS. The blood is added immediately before extraction. The standard procedures of extraction, chromatography and detection of spots are used. See text for abbreviation.

262 TABLE

IV

EXTRACTION OF AMNIOTIC NOL (2 : 1, V/V) With the exception

FLUID

of the extraction

WITH

DIFFERENT

volumes

the standard

VOLUMES procedures

OF CHLOROFORM~METHAas described

under

methods

are

used. _ peak area (arbitrary

units)

Interphase

Volume (ml) chloroformmethanol added to 2 ml centrifuged amniotic fluid -

Scanning -_Lysophosphatidylcholine

Sphingomyelin

Phosphatidylcholine

Phosphatidylethanolamine

2 5 10 15

<3 11 25 17

<3 ‘7 7 9

22 43 44 31

15 I3 16 7

---_.

Broad Broad Thin Thin

extraction of the upper- and interphase after primary shaking with chloroformmethanol in the proportion of 1: 5, no further detectable amount of P-lipids can be extracted. I~f~~e~ce of the euaporatio~ temperut~re on the ~-~i~id~ L/S is lower by evaporation of the chloroform phase at 40” in 20 min than at 70” in 20 min (Table V). In another experiment with evaporation of another AF in 5 min at 40” and 70” no alterations in the P-choline fractions could be detected. Studies on the optimal conditions for development of the molybdenum biue colour The development of the molybdenum blue colour is maximal after incubation for 15 min at loo”, for all P-lipid fractions with the exception of P-ethanolamine, where maximal colour development can be traced only after more than 15 min incubation at 110” (Figs 3-4). Higher temperatures than 110” or longer heating time than 15 min decreases the colour intensity. The colour is stable for about 48 h in the dark.

TABLE

V

L/S AND EVAPORATION

TEMPERATURE

9 times 2 ml centrifuged amniotic fluid extracted with chloroform--methanol (2 : 1, v/v) for 5 min and evaporated in a stream of nitrogen in water bath for 20 min at the temperatures indicated. The standard procedures of chromatography and detection of spots are used. See text for abbreviations. Evaporation temperature 40 55 70 -.

f”) mean of three samples mean of three samples mean of three samples

L/S fmoles/mofe)

LysoiS fmoles/mole)

9.5 + 0.8 11.1 k 0.9 12.2 k 1.0

4.4 If: 0.4 3.8 + 0.4 4.1 + 0.5

263

70. ___._50_

&__________-__-__---*-----t

E :50-AI___________-_--------*-?6 .’

-

*____________________+_ 0

lo-*--

I:

-2 loI

0

5

Scanning

a__ a-

E =30 &__________--__----.-------: )e

40-w

time

10 after

15 20 heating I” wen

25

30 Hours

_,Q_ ..___ ___________________.__ 0 _..___-_%-,,,.,....,.,.,,....,,,,,,..,,, 5 Scanning

IO

tlmeaftcr

15 heatmg

20 I” oven

25

30 HCUE

Figs. 3-4. The scanning peak area of the four amniotic fluid P-lipids after spraying with ammonium molybdate. at different times after heating in oven for different periods of time and at different temperatures. l-ml portions of centrifuged amniotic fluid are used. Extraction, chromatography and detection of spots are performed as described under Methods. (The standard deviation of the single P-lipid peak area is maximally 15% on one-dimensional TLC, 10 double measurements). o-0: 15 min heating in oven at 100°. A-A: 15 min heating at 110’. O-0: 10 min heating at lOO’.--in Fig. 3: Lysophosphatidylcholine. in Fig. 3: Sphingomyelii. -- in Fig. 4: Phosphatidylethanolamine. in Fig. 4: Phosphatidylcholine.

Studies on correlation of data obtained by assay of phosphorus fractions with those obtained by direct scanning

content

in TLC

Accepting a normal distribution of data, regression analysis revealed a linear relation between phosphate content and P-lipid content expressed by the scanning peak area after spraying with ammonium molybdate for the four P-lipids examined (Tables VI -1X). It will be permissible to estimate L/S 1 h after ammonium molybdate spraying as the regression coefficients are approximately identical with Pcholine and Sph. Colour development induced by small quantities of P-ethanolamine (0.012 pmoles P), lyso-P-choline (0.006 pmoles P) and Sph (0.005 pmoles P) (Tables IX, VI, and VII) is finished after 1 h. Consequently after 1 h it is possible to use the proportions of peak areas of P-ethanolamine or lyso-P-choline to that of Sph as a marker of changes in the amounts of these lipids. The molar ratios are designated E/S X h1 and Lyso/S X k2 where k, and k2 are constants depending on the proportions of the regression coefficients for P-ethanomaline-Sph and lyso-P-choline-Sph (Tables IX, VII, and VI). AF ~01s. of about 0.63 ml often correspond to the applied quantity of lipids giving rise to complete colour development during a period of 1 h. Recovery

analysis

Using the standard procedure for lipid extraction and P-determination a recovery was found of 103.5% f 5% (double measurements) expressed in per cent of added P-choline phosphate. The standard deviation of the data obtained by the one-dimensional direct TLC method was for L/S 8%, for Lyso/S lo%, and for E/S 12% (10 double measurements).

VI

ANALYSIS

-

THE LYSOPHOSPHATIDYLCHOLINE

FRACTION

0.026 0.015 0.006 0.001

2.50 1.25 0.63 0.31

* The phospholipids are in all 8 cases extracted tion and chromatography axe used.

Phosphate in the Iysophosphatidyleholiine fraction on chromatopiate (f..imolest

Amniotic fluid centrifuged (ml)*

contents = ordinate,

with

37 22 17 13 r = b = rt P <

44 24 19 12 r = 0.98 b = 0.0008 + 0.0001 P
lh

10 ml chloroform--methanol

0.98 0.0010 0,0001 0.01

5 min

(2

: 1. vi\) in a whirlmixer

24 18 14 F = 0.36 b = 0.0007 ci: 0.0001 P <0.0125

49

2h

0.98 0.0006 0.0001 0.01

-

---.-

nf exapora-

53 2S 17 15 I’ = 0.98 b = 0.0006 t 0.0001 P
25h

..----_

= 0.07) in arbitrary

for 5 mill. The standard procrdurcs

55 30 22 16 r = b = c P <

19.5 h

TLC (RF-value

the scanning peak area = abscissa.). P = the level

Scanning peak area of lysopho~h~tidylcho~ne spot on one-dimensional units (molybdenum biue), different time after heating in oven -___---____

r = correlation coefficient. b = regression coefficient (the slope of the curve). (The phosphate of significance for b. (Olivetti Underwood programa 101. Regression analysis code 3.40).

REGRESSION

TABLE

TABLE VII

0.014 0.010 0.006 0,000

2.50 1.25 0.63 0.31

5 4 3 3 r = 0.93 b = 0.0059 f 0.0012 P
5 min 8 5 4 3 r = 0.93 b = 0.0026 +i 0.0005 P KO.025

lb

(2

0.95 0.0019 0.0003 0.025

5 2 r = 0.96 b = 0.0017 f 0.0002 P
6

10

19.5 h

12 5 3 2 r = 0.89 b = 0.0012 0.0003 4 P x0.05

25h

: 1, v/v) in a whirlmixer for 5 min. The standard procedures of evapora-

5 4 2 r = b = f P<

8

2h

Scanning peak area of sphingomyelin spot ou one-dimensional TLC (RF value = 0.14) in arbitrary units (molybdenum blue). different time after heating in oven

FRACTION

* The phospholipids are in all 8 cases extracted with 10 ml chlorofonw-methanoi tion and chromatography are used. Abbreviations: see Table VI.

Phosphate in the sphingomyeiin fraction on cbromatoplate (j.&nOl.sS)

Amniotic fluid centrifuged (ml)*

REGRESSION ANALYSIS - THE SPUINGOMYELIN

VIII

146 108 61 45 r = b = + P <

127 94 48 46 r = 0.98 b = 0.0028 It 0.0003 P
see Table VI.

with 10 ml chloroform-methanol

lh

5 min

are used. Abbreviations:

are in alI 8 cases extracted

tion and chromatography

* The phosphotipids

0.271 0.132 0.054 0.024

2.50 1.25 0.63 0.31

FRACTION

(2

: 1, v/v) in

0.98 0.0022 0.0002 0.005

a whirlmixer

158 112 57 52 i- = b = t P <

2h 173 134 73 52 r = b = L P <

of evapora-

185 158 61 59 r = 0.93 b = 0.0015 0.0003 k 1’<0.025

25h

value = 0.25) in arbitrary

0.97 0.0019 0.0003 0.01

19.5 h

TLC (RF

for 5 min. The standard procedures

Scanning peak area of phosphatidylcholine spot on one-dimensional units (molybdenum blue), different time after heating in oven

- THE PHOSPHATIDYLCHOLINE

Phosphate in the phosphatidylcho~ne fraction on chromatoplate (/.knoles)

ANALYSIS

Amniotic fluid centrifuged (ml)*

REGRESSION

TABLE

40 18 12 8 r = b = k P< 1.00 0.0013 0.00903 0.0006

Smin

0.99 0.0008 0.00005 0.0026

0.98 0.0005 0.00005 0.005

(2 : 1, v/v) in a whirlmixex for 5 min. The standard procedures of evapora-

0.99 0.0005 0.00003 0.0026

87 39 I.3 12 r = b = f; P<

86 35 16 13 F= b = * P<

57 24 13 11 r = 0.99 b = 0.0009 t 0.00005 P < 0.0026

59 28 14 9 r = b = + P<

in

25 h

0.37)

19.5 h

=

2h

value

lh

* The phospholipids are in alI 8 cases extracted with 10 ml chloroform-methanol tion and chromatography are used. Abbreviations: see Table VI.

0.047 0.018 0.012 0.005

FRACTION

Scanning peak area of phosphatidylethanolamine spot on one-dimensional TLC (RF arbitrary units (molybdenum blue), different time after heating in oven ._.

PHOSPHATIDYLETHANOLAMINE

2.50 1.25 0.63 0.31

-THE

Phosphate in the phosphatidylethanolamine fraction on chromatoplate (E(moIes)

ANALYSIS

Amniotic fluid centrifuged (ml)*

REGRESSION

TABLE IX

268

Discussion Illvestigators [16-20] have found different, correlations between L/S and development of RDS. These differences are connected with lack of agreement on criteria for defining the syndrome and with chemical assay conditions. L/S is used as a measure for the P-choline content in the AF because the volume in which P-choline has been dissolved is unknown. L/S after precipitation with cold acetone is used as a measure for the lung maturity. Thus Gluck [S] has proved that the surface-active P-cholines are precipitated with cold acetone, that the Sph production is relatively constant in proportion to the P-choline production [5], that the Sph concentration is higher than the P-choline concentration until the 27th week of gestation [5], and that P-choline and Sph concentrations were nearly equal prior to the 35th week when the P-choline concentration rose sharply. Gluck also postulated a complete precipitation of Sph with cold acetone. L/S is simpler to use than quantitative determination of the single P-lipid. Ratios for the acetone-precipitated P-lipids are occasionally higher than for the non-precipitated ones [6], and may be a consequence of uncomplete precipitation of Sph, and for the lyso-P-choline group a consequence of P-choline decompositioi~ caused by acetone. Higher L/S after acetone precipitation has been observed by other investigators [ 191. By following the molybdenum blue colour development for several hours after heating, an increase was found in the peak area for all four P-lipids investigated, most pronounced in the major fractions. This might be owing to the fact that the reduction of molybdate is initiated at the sites of double bonds present in the unsatiated fatty acids ill]. Un~turated fatty acids are present even in the acetone-precipitated P-lipids. Thus Gluck et al. ]5] found 19.4% unsaturated a-fatty acids, and 20.4% unsaturated p-fatty acids in acetone precipitated P-choline from AF at the term. The present investigations favour a staining time of 1 h, since at this time P-choline and Sph have caused similar development of the molybdenum blue colour per mole P-lipid. After sampling of AF phospholipases will deacylate P-choline by standing. As shown in the present communication this can be avoided by direct extraction, by deep freezing or by adding EDTA, consequently Ca*’ is presumed to be a co-factor for the enzyme [ 151 . Contamination with bacteria, mucus, meconium or blood may influence the results. This paper also reveals that L/S depends on the proportion of the chIoroform-methanol added, and on the evaporation temperature. Thus, by evaporation at 40” for 20 min, a lower L/S than .by evaporation at 70” can be seen. This could be owing to decomposing substances in the bottom phase. However, this process is inhibited at 70”. A similar phenomenon was discovered in extraction of plasma [21]. Evaporation at 70” with a fast stream of nitrogen is therefore recommended. Acknowledgement This work is supported by grants from Ingemann 0. Bucks fond, Odense, and “Den laegevidenskabelige fond for St@kobenhavn, Faer@erne og Grfinland”.

269

References 1 A. Patz. Am. J. Ophthalmol., 38 (1954) 291. 2 A.J. Schaffer and E.M. Avery. Diseases of the Newborn. (3rd edn.), Saunders. Philadelphia-LondonToronto, 1971, pp. 90 and 93. 3 E.M. Scarnelli, Adv. Pediat.. 16 (1969) 177. 4 L. Gluck and M.V. Kulovich, Ped. Clin. North Am., 20 (1973) 367. 5 L. Gluck. M.V. Kulovich. R.C. Borer, P.H. Brenner. G.G. Anderson and W.N. Spellacy. Am. J. Obstet. Gynec., 109 (1971) 440. 6 H. Verder and J. Clausen. Clin. Chim. Acta, 51 (1974) 271. 7 J. Folch, M. Lees and G.H. Sloane Stanley, J. Biol. Chem., 226 (1957) 497. 8 L. Gluck. E.K. Motoyama, H.L. Smits and M.V. Kulovich. Pediat. Res.. 1 (1967) 237. 9 E. Stahl. Dtinnschicht-Chromatografie, Springer, Berlin-Gottingen-Heidelberg, 1962. P. 7. 10 H. Wagner, L. Hdrhammer and P. Wolff, Biochem. Z.. 334 (1961) 175. 11 H.O. Christensen Lou, J. Clausen and F. Bierring. J. Neurochem.. 12 (1965) 619. 12 V.P. Whittaker and S. Wijesundera. Biochem, J., 51 (1952) 348. 13 V.P. Skipski, R.F. Peterson and M. Barclay, Biochem. J., 90 (1964) 374. 14 C.J.F. Bijttcher. C.M. van Gent and C. Pries. Anal. Chim. Acta, 24 (1961) 203. 15 R.M.C. Dawson and N. Hemington, Biochem. J.. 102 (1967) 76. 16 J. Nakamura, J.F. Roux, E.G. Brown and A.Y. Sweet, Am. J. Obstet. Gynec., 113 (1972) 363. 17 J.D. Schulman, J.T. Queenan. E.M. Scarpelli. E. Church and P.A.M. Auld, Obstet. Gynec., 40 (1972) 697. 18 D. Armstrong and D.E. Van Wormer, Am. J. Obstet. Gynec., 114 (1972) 1083. 19 L. Sarkozi. H.N. Kovacs, H.A. Fox and T. Kereneyi. Clin. Chom., 18 (1972) 956. 20 L. Gluck and M.V. Kulovich, Am. J. Obstet. Gynec.. 115 (1973) 539. 21 0. Vikrot, Acta Med. Stand.. 175 (1964) 443.