133

Clinica 0

Chimicn

Acta,

74 (1977)

Elsevier/North-Holland

133-138

Biomedical

DETERMINATION CONCENTRATION LUNG MATURITY

OF AMNIOTIC FLUID PALMITIC ACID FOR THE ESTIMATION OF FETAL

A. BICHLER

a,*, C:. DAXENBICHLER

a Dfparlmetlt

of

lIocumftilatiori,

(Received

July

Press

Obstetrics

and

I’rficvrsily

of Inr~sDruch.

Zlst,

a and

Gynecology

W. GEIR and

b

h Ucparlmcul

Inn.sbritck

of’l~iostotistics

and

(rlusfria)

1976)

Summary Palmitic acid concentrations in amniotic fluid (AF) were determined in 135 patients with normal and pathological pregnancies between the 27th and 42nd week of gestation. There was a sharp rise in the mean palmitic acid concentration after the 34th weeks of gestation from 2.7 pg/ml to 9.9 pg/ml at term. This increase is almost identical with the rise of AF-lecithin. It was found that between 70% and 100% of AF-palmitic acid originates from lecithin. 65 patients were delivered within 24 h after amniotic fluid sampling. 7 infants of these patients developed a respiratory distress syndrome (RDS). In all cases with RDS AF-palmitic acid concentration was far below 5 pg/ml. Assuming an AF-palmitic acid concentration >5 pg/ml for characterising fetal lung maturity (= no RDS), there were no false negative results, but 16% false positive results. However, the determination of AF-palmitic acid concentration seems to be a most reliable method for the assessment of fetal lung maturity.

Introduction In recent years a relationship between amniotic fluid phospholipid content and fetal lung maturity has been shown [l-4]. It has been demonstrated that lack of lecithin is an important factor in the development of the respiratory distress syndrome (RDS) in newborns [ 21. Meanwhile several methods for estimating phospholipids in the amniotic fluid have been developed [ 1,2,4-7,161. At present the most common method is the determination of the lecithin-sphingomyelin (L/S) ratio according to the method of Gluck and coworkers [ 2,6]. It is well known that dipalmytoyl lecithin (DPL) is the major surfactant of the * Correspondence Austria.

to:

Dr. A. Bichler,

UniversitLts-Frauenklinik.

6020

Innsbruck,

Anichstrassr

35,

134

alveolar lining layer [S] . It is synthesized by the phosphocholine transferase pathway between the 34th and 36th week of gestation when the fetal lung becomes mature [ 91. Therefore the estimation of DPL in the amniotic fluid ought to be a most reliable method in the assessment of fetal lung maturity. Warren and coworkers found that roughly 90% of the total palmitic acid in the amniotic fluid originates from lecithin. [lO,ll]. They found a sharp rise of the concentration of palmitic acid in the 35th week of gestation. The values found in infants with mature lungs were much higher than those obtained in newborns with RDS. However, there exist at present no exact data on the critical limiting values which make the prediction of an RDS possible [lo---12). The purpose of this paper is to define these values by more clinical results. Material

and method

135 amniotic fluid specimens were collected from patients with normal and pathological pregnancies between the 27th and 42nd weeks of gestation. The samples were obtained at amniotomy, by amniocentesis or at the time of caesarean section. Samples contaminated with blood or meconium were discarded. Specimens not processed immediately were stored frozen at -24°C until analysis. of palmitic acid in the amniotic fluid The samples were centrifuged at 1500 X g for 10 min. The amniotic fluid should be perfectly clear; if lumps of vernix remain in the supernatant, filtration is necessary; otherwise the palmitic acid concentration will be increased. The extraction was performed by the method of Folch [13] with slight modifications: 1 ml of amniotic fluid was mixed together with 1 ml of methanol and the mixture was extracted twice with 3 ml of chloroform. After evaporation of the solvent in a stream of N, at room temperature, the phospholipids of the residue were hydrolysed with 0.5 ml sodium hydroxide in methanol (0.5 ml) for 10 min at 60°C. Methylation of the free fatty acids was assessed according to the method of Metcalfe [ 141 by adding 1 ml of BF, in methanol to the hydrolysis mixture and by heating for 10 min at 60°C. After methylation, 2 ml of a saturated NaCl solution were added and the mixture was extracted once with 5 ml of petroleum ether (b.p. 4060°C). The solvent was dried with 0.1 g of sodium sulfate, evaporated in an N, stream and the residue redissolved in 0.3 ml of Ccl,. 2 1.11of the extract were injected into a gas chromatograph equipped with a 2-m glass column and an FID. OV-225 respectively OV-1 were used as stationary phases; the oven temperature was 150°C; the temperature of injector block and FID was 200°C. The quantitative estimation was done by comparing the peak height of the sample with the peak height of a palmitic acid methyl ester standard. Fig. 1 shows a gas chromatogram of a methylated extract of amniotic fluid. The recovery for each step was found to be over 9070, the total recovery was 81% on average. Dipalmitoyl lecithin (Serva, D-69 Heidelberg, G.F.R.) served as a reference substance. The standard deviation was calculated by performing 20 determinations out of an amniotic fluid pool and was found to be ?7%.

Determination

135

1 2

Gas-chromatcqam fluid

extract, the

in this

paper.

I : :

Peak

Peak 2

of an amniotic which

following

-I

was treated

procedure

described

solvent

KC141

Palmitic

acid

methyl

ester

18G ng injected1 Gaschromatqra;llhic

2m

x 3 mm

containing Carrier

:

Lsnditions glass

column

3% OV 225 on 2schrom gas IN21 fiowrate

Injector temperature Fig.

I.D.

temperature 15O’C;

200°C, FID

0.

30 ml:min. column

temperature

2oO°C.

1.

The specificity of the method was proved by a mixture of the 10 most common fatty acid methylesters. It was found that on OV-225 as well as on OV-1 palmitic acid methylester was quantitatively separated from the other methylesters. Another indicator of specificity was the identity of the values obtained on OV-225 and OV-1. Results There is a sharp rise in the mean AF-palmitic acid concentration after the 34th week of gestation from 2.7 pg/ml to 9.9 pg/ml at term (Fig. 2). In a subsequent paper correlations will be reported between the concentration of palmitic acid and those of some other phospholipids in the amniotic fluid. There was a medium correlation between palmitic acid concentration and L/S ratio (r, = 0.6387, p < 0.05).

AF-palmitic acid concentration 65 patients were delivered

and the incidence

of RDS

within 24 h after amniotic fluid sampling. developed an RDS, 5 of these died from Hyaline

7 infants of these patients Membrane disease (HMS). Fig. 3 shows a plot of palmitic acid values in these patients. A palmitic acid concentration over 5 pg/ml was considered to mark fetal lung maturity. In 48 out of 57 newborns (84.2%) without RDS, lung maturity was predicted correctly (palmitic acid concentration >5 pg/ml). In 9 cases (15.8%) there was a

Palmitic acid liJglml~

I

I

I

I

I

I

I

21-M

31-32

33-34

35-36

37-38

39-40

11-42 week of gestation

Palmitic acid concentration in the amniotic fluid during gestation imedian with fhe confidence limits, p

Determination of amniotic fluid palmitic acid concentration for the estimation of fetal lung maturity.

133 Clinica 0 Chimicn Acta, 74 (1977) Elsevier/North-Holland 133-138 Biomedical DETERMINATION CONCENTRATION LUNG MATURITY OF AMNIOTIC FLUID P...
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