Phospholipid Surface Tension - Clinical Chemistry

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Mar 19, 1988 - distilled water, and 5 mL of perchloric acid (700 g/L), and stored this at 4 ... transferred to a 15-mL glass-stoppered centrifuge tube, vortex- ..... Ruiz J. Estudiodela concentraci#{243}ndelosfosfolpidosenel lIquido amni#{244}tico.
CLIN. CHEM. 35/5, 800-803 (1989)

Phospholipid Surface Tension: the Diagnostic Utility of Amniotic Fluid and Its Lipid Extract, an Analysis of the Value of Precipitation with Cold Acetone J. Rulz

Budrfa,1J. M. Abbad Bara,1 E. Fabre Gonzalez,2 M. T. Hlgueras Sanz,2and J. L Serrano Ost#{225}rlz1’3

In prevention of the respiratory distress syndrome (ADS), measurements of surface tension values (a biophysical property) of amniotic-fluid samples are correlated with their lecithin/sphingomyelin (US) ratios (a biochemical property). According to some authors, precipitation of phospholipid with cold acetone is essential for determining the US ratio, because it separates surfactant and nonsurfactant phospholipidic fractions. Here we report the first study of the ability of three amniotic-fluid components to decrease surface tension: The complete lipid extract (without precipitation), and the fractions precipitated and (or) remaining soluble after addition of cold acetone. Addition of increasing aliquots of lipid extracts to these three samples showed that: (a) measurement of surface tension rapidly and reliably indicates fetal lung maturity, and (b) both precipitated and soluble phospho-

lipid fractions decrease surface tension similarly, making it unlikely that the precipitation step in fact separates surfactant and nonsurfactant material.

AddItIonalKeyphrases:fetal status tio

lecithinlsphingomyelin ra-

chromatography,thin-layer

In respiratory distress syndrome (RDS), deficiency of surface-tension-lowering substances (surfactant material) in alveoli of the newborn infant is the primary etiologic factor.4 The ability to predict this deficiency prenatally would consequently decrease the incidence of RDS and improve neonatal survival. The efficiency of amniotic-fluid

analysis

for the documen-

tation of fetal lung maturity has become generally accepted (1, 2). Two general types of assays are generally used to measure amniotic-fluid surfactant: biochemical quantification and biophysical measurements. All current biochemical methods for evaluating lung maturity involve estimating the concentration in amniotic fluid of phospholipids, considered the main components of pulmonary surfactant (3), as a reflection of the surfactant

produced by the fetus. In 1967, Gluck et al. (4) showed that surfactant and nonsurfactant phospholipids could be separated by precipitation with cold acetone. From the surface-tension measurements of the different phospholipids obtained from both fractions, these authors deduced that lecithin (L), sphingomyelin (5), phosphatidyldimethylethanolamine, and phosphatidylinositol had surfactant activity, whereas phosphatidylethanolamine and phosphatidylserine did not. Later, Gluck et al. (5) proposed a new diagnostic method for fetal lung maturity, based on the L/S ratio determination obtained after precipitation with cold acetone. This method Departments of’Organic Chemistry and2 Obstetrics & Gynecology, University of Zaragoza, 50009 Zaragoza, Spain. 3Author for correspondence. Nonstandard abbreviations: RDS, respiratory distress syn-

drome;L’S, lecithin/sphingomyelin

ratio; TLC, thin-layer chroma-

tography; and HP-, “high-performance.” Received March 19, 1988; accepted February 27, 1989.

800 CLINICALCHEMISTRY, Vol. 35, No. 5, 1989

has been widely accepted, although there is still controversy as to whether the precipitation step should be included (68).

The surfactant capacity of amniotic fluid can also be defined by measuring its physical effect on alveolar cells, i.e., the decrease in surface tension. In 1973, Shelley et al. (9) used diagrams of surface-tension area to show that amniotic fluid taken at delivery of newborn infants who later developed RDS had little or no surfactant activity, and that these results correlated well with L/S ratios. Mueller-Tyle et al. (10) used a Wilhehny balance and observed a decrease in amniotic-fluid surface tension as gestational age increased. Goldkrand et al. (11, 12) proposed a diagnostic method for fetal lung maturity based on the decrease in surface tension in the lipid extract obtained from amniotic fluid without the cold-acetone precipitation step; they reported a good correlation between these properties of the amniotic-fluid lipid extract, the L’S ratio, and the probability of neonatal RDS. Frosolono and Roux (13) carried out similar work but used amniotic-fluid and pulmonary tissue. For this study, we analyzed the surface-tension values of centrifuged amniotic-fluid samples and of lipid extracts obtained from the same samples. We correlated these values with the US ratios for the same samples, in an attempt to establish a comparison between the biochemical and biophysical properties of surfactant material in amniotic fluid. We also compared the capacity of lipid extracts obtained from amniotic-fluid samples, with and without cold-acetone precipitation, to decrease surface tension.

Materials and Methods Materials TLC plates: We used 10 x 10 cm silica gel F2M “highperformance” thin-layer chromatographic plates (HPTLC) (Merck, Darmstadt, F.R.G.), especially designed for nanoscale chromatography. Equipment: Centrifuge: Hettich Rotanta K centrifuge

(Hettich, Tuttlingen, F.R.G.). Chromatography injector: Nanomat 2770-1078-E with applicator 27750-278-E (Camag, Muttenz, Switzerland). Chromatography tank: Camag twin-trough chamber for HPTLC. Densitometer: Camag TLC scanner light source, equipped with an integrator AIM 65 analogic digital interface (Rockwell, Muchen, F.R.G.). Surface-tension balance: Kruss interfacial-tensiometer K8 with Pt-fr ring (Kruss, Hamburg, F.G.R.). Chromatographic solvent: Chloroformlhexane/methanoll glacial acetic acid (5/3/1.6/1 by vol) (14).

Staining reagent: We dissolved 5 g of phosphomolybdic acid in a mixture consisting of 25 mL of ethanol, 70 mL of distilled water, and 5 mL of perchloric acid (700 g/L), and stored this at 4 #{176}C until required. Amniotic-Fluid Samples Forty-nine specimens of amniotic fluid were obtained by amniocentesis or by transvaginal sampling

transabdominal

after artificial amniorrhexis, between 28 and 42 weeks of

gestation. Amniocentesis was performed for medically indicated reasons when fetal lung status might influence management. All patients underwent ultrasonography before the procedure. Meconium- and blood-stained fluids were discarded. If the sample could not be used on the day collected, it was stored at -20 #{176}C until required. All patients involved in this study were cared for by the Zaragoza University Clinical Hospital Perinatal Services. Procedures Phospholipids art raction: The amniotic-fluid samples were centrifuged at 2500 x g for 5 mm to remove cellular debris. The supernate was divided into two aliquots, which were processed simultaneously. The first aliquot (2 mL) was transferred to a 15-mL glass-stoppered centrifuge tube, vortex-mixed with an equal volume of absolute methanol for 15 s, then combined with 4 mL of chloroform and vortexmixed for about a minute. The emulsion formed was dispersed by centrifugation at 1500 x g for 5 min. Three layers were formed in the tube: an aqueous upper layer, a protein layer at the interface, and the chloroform (lower) layer, which contained the lipids. We transferred the chloroform lay or to a conical tube and evaporated the solvent under a stream of nitrogen in a water bath at 60#{176}C. This lipid extract was designated the “complete” fraction (Fraction A). The secondaliquot (2 mL) was processedsimilarly and the lipid extract residue was redissolved in a minimal amount of chloroform. We added excess cold acetone dropwise until no more white precipitate was formed and kept the sample in a bath of crushed ice for 15 mm. We then centrifuged the sample at 1500 x g for 5 mm, decanted the supernate into another conical tube, and evaporated the solvent from both the supernate and the precipitate, under a stream of nitrogen at 60#{176}C. The lipid extracts obtained were designated “precipitated” (P) and “soluble” (S) fractions. Surface-tension measurements: We measured surface tension in amniotic-fluid samples that had been centrifuged (2500 x g, 5 mm; unprocessed fraction), and in the A, P, and S fractions of amniotic-fluid lipid extracts. For the unprocessed amniotic-fluid samples we used an interfacial tensiometer (Model K-8; KrUss) fitted with a platinum-iridium ring. The balance was calibrated daily by measuring the surface tension (72.6 dynlcm at 20 #{176}C) of doubly distilled water. The surface tension of fractions A, P, and S was measured as follows. Each dried residue was dissolved in 2 mL of chloroform and kept on crushed ice (to prevent the chloroform from evaporating). We ifiled a glass dish (50 mm diameter) with 10 mL of doubly distilled water, submerged the Pt-b ring in the water, and layered 25 L of the reconstituted lipid extract onto the water surface, using a

microliter syringe (Hamilton, Bonaduz, Switzerland; cat. no. 705N). We allowed all visible droplets to spread over the water surface before we slowly withdrew the ring and measured the surface tension. We repeated the measurements by adding another 25-pL aliquot of the extract, up to a total volume of 200 pL of lipid extract added, each time submerging the ring before repeating the measuring procedure. The US ratio determination: We measured the US ratio in the lipid extracts at the same time as the surface tension measurements, using the method described by Gluck et al. (5), as modified by Fabre et al. (14). Statistical methods: All data were analyzed and are reported as means ± SEM. To establish significant or nonsignificant differences, we compared results by using analysis of variance

(15).

Results and Discussion Surface-Tension

Reproducibility

We assessed the precision of the method by measuring the surface tension of various aliquots (from 25 to 200 L added) of chloroform solutions containing 0.11 g of amniotic-fluid phospholipid extract per microliter (110 mg/L) during five

consecutive days. As Table 1 shows, within-run and between-run CVs were