Amniotic Fluid Phospholipids Measured by ... - Clinical Chemistry

CLIN.CHEM.26/3, 403-405 (1980)
Amniotic Fluid Phospholipids Measured by Continuous-Development
Thin-Layer Chromatography
Mark D. Kolins, Emanuel Epstein, W. Harold Civin, and Sheldon Weiner’
We describe a one-dimensional thin-layer chromatographic
system for separation of amniotic fluid phosphatidyicholine,
sphingomyelin, phosphatidylglycerol, phosphatidylinositol,
phosphatidylserine, and phosphatidylethanolamine
in amniotic fluid. We utilize short-bed continuous development
and “high performance” thin-layer chromatography.
Phospholipids are detected with an antimony molybdate
staining reagent and quantitated by transmittance
densitometry. This system is more sensitive to changes
in lecithin/sphingomyelin ratios than are planimetric
evaluations.
AddItIonalKeyphrases: lecithinlsphingomyelin ratio fetal
status
Amniotic fluid phospholipids are currently assayed as an
aid in predicting fetal lung maturity. The measurement most
commonly used in the clinical laboratory is the ratio of
phosphatidylcholine (lecithin) to sphingomyelin. Although
use of the lecithin/sphingomyelin (L/S) ratio is satisfactory
when applied to normal pregnancies, its predictive value is less
certain in pregnancies complicated by maternal diabetes
mellitus or toxemia (1, 2). This fact has led to investigations
of the potential use of other amniotic fluid phospholipids in
predicting fetal lung maturity, and phosphatidylglycerol assay
is now considered to be important in predicting fetal lung
status (2-4). In addition, in cases of respiratory distress syndrome,
the proportions of phosphatidylserine and phosphatidylethanolamine
increase. The assay of these two phospholipids
also may be helpful in distinguishing the mature and
immature fetal lung (3).
The one-dimensional chromatographic systems (4, 5) for
obtaining L/S ratios and relative percentage of phosphatidylglycerol
fail to resolve phosphatidylserine and phosphatidylethanolamine.
In current clinical and investigtional
laboratory methods for individual quantitation of major
amniotic fluid phospholipids, two-dimensional thin-layer
chromatography is used.
We describe here a short-bed continuous-development
technique of “high-performance” thin-layer chromatography
in one dimension, with which we can distinguish the major
phospholipids of amniotic fluid (phosphatidylcholine,
sphingomyelin, phosphatidylinositol, phosphatidylserine,
phosphatidylethanolamine, and phosphatidyiglycerol).
Materials
and Methods
Reagents and Apparatus
We obtained egg lecithin/bovine sphingomyelin mixtures
in weight ratios of 1/1, 2/1, 3/1, 4/1, and 5/1 from Applied
Science Labs., Inc., State College, PA 16801. Bacterial phosphatidylglycerol
and bovine phosphatidylethanolamine were
obtained from Supelco, Bellefonte, PA 16823. Porcine brain
Departments of Clinical Pathology and 1 Obstetrics and Gynecology,
William Beaumont Hospital, 3601 West Thirteen Mile Rd., Royal
Oak, MI 48072.
Received July 27, 1979; accepted Dec. 4, 1979.
phosphatidylserine and porcine liver phosphatidylinositol
were supplied by Miles Laboratory, Elkhart, IN 46514. We
used Merck Silica Gel 60 high-performance thin-layer chromatographic
plates (10 X 10 cm) from Scientific Products,
Romulus, MI 48174; an ACD-18 automatic computing densitometer
from Gelman Instrument Company, Ann Arbor, MI
48106; and a short-bed continuous-development chamber
from Regis Chemical Co., Morton Grove, IL 60053. The mobile
phase consisted of petroleum ether (boiling point range 20-40
oC)/chloroform/methanol/acetic acid (3/5/1.5/1 by vol).
We used an antimony molybdate phosphate reagent (6) to
stain phospholipids, consisting of a 10/1/3/6 (by vol) combination
of 2.52 mol/L sulfuric acid, and the following aqueous
solutions: 8.2 mol/L antimony potassium tartrate, 32.3
mmol/L ammonium molybdate, and 0.1 molfL ascorbic acid.
This reagent mixture is stable for 28 days at 4 #{176}C. We combined
8 mL of this mixture with 1 mL of isopropanol and 13
mL of distilled water to prepare the active staining reagent,
which must be used promptly.
Method
Amniotic fluid specimens were obtained by transabdominal
aspiration between 30 and 42 weeks of gestation. Fluids were
obtained at cesarean section by needle aspiration before
rupture of the amniotic membranes. Meconium- and bloodstained
fluids were discarded.
Acetone-precipitable phospholipids were isolated from
amniotic fluids as described by Gluck et al. (7). The isolated
phospholipids were dissolved in 20 tL of chloroform, and 10
was applied to the high-performance thin-layer chromatography
plates in streaks about 1 cm long. The plates were
placed at a reduced pressure in a desiccator-oven at room
temperature for 5 mm. The mobile phase was placed in the
short-bed continuous-development chamber and the plates
were positioned to give the maximum bed length. Plates were
developed for 50 mm, then blown dry with cool air from a hair
dryer. The antimony molybdate phosphate reagent was
poured over the plate and left at room temperature for 1 mm.
The stained plates were dried with warm air from a hair dryer
for 5 mm to develop the phospholipid bands. Phospholipids
were identified by migration identical with standards. Each
phospholipid was measured by transmittance densitometry
at 620 nm and expressed as a percentage of total phospholipids.
Results
AnalyticalVariables
Separation of phospholipids. We could clearly separate all
six of the major phospholipids of amniotic fluid with this
system (Figure 1).
Linearity. Linearity was assessed by utilizing commercially
prepared lecithin/sphingomyelin mixtures. Both our procedure
with use of densitometry and the method of Gluck et al.
(10) with planimetry gave linear results for L/S weight ratios
of 1 through 5. The ratios by densitometry showed a greater
response to changes in the lecithin and sphingomyelin weight
mixtures (Figure 2).
CLINICALCHEMISTRY,Vol.26, No. 3, 1980 403

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8
7
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Fig. 1. Separation of the amniotic fluid phospholipids from amniotic
fluid obtained at cesarean section
S, sphingomyelin; L, lecithin; P1,phosphatidylinositol; PS,phosphatidylserine;
PE. phosphatidylethanolamIne; P0, phosphatidylglycerol; X, unidentifIed
Color stability. The phospholipids appear as blue bands
within 5 mm. The color is stable for at least 30 mm.
Accuracy and precision. Commercial mixtures of phosphatidylcholine
and sphingomyelmn at weight ratios of 1/1,2/1,
3/1,4/1, and 5/1 were assayed by the described method. Corresponding
L/S ratios in duplicate determinations were 1.30
and 1.38, 1.97 and 1.93, 2.27 and 2.33, 3.08 and 3.06, and 3.98
and 3.82.
We assessed within-day precision for the high L/S ratio
range by running 10 aliquots of pooled amniotic fluid obtained
at cesarean section. The mean L/S ratio was 9.13 (SD 0.88),
with a CV of 9.68%. The mean percentage of phosphatidylglycerol
(of total phospholipid) was 3.23 (SD 0.59), with a CV
of 18.4%. The mean percentage of phosphatidylmnositol was
26.3 (SD 2.7), with a CV of 10.2%.
Within-day precision for the low L/S ratio range was assessed
by isolating 10 aliquots of an equi-weight mixture of
phosphatidylcholine and sphingomyelin. The mean L/S ratio
ss
Fig.2.Densitometrictraceofamnioticfluidphospholipids
Abbreviations as in Fig. 1
P1
-‘-
L
1
Borer’s
Method
HP-TLC
Fig.3.Distribution ofLIS ratios inpairedspecimens runbythe
presentmethod (HP-TLC)and thatofGlucketal.(10) (Borer’s
method)
was 0.85 (SD 0.08), with a CV of 9.56%. Day-to-day precision
was assessed by isolating phospholipids on four consecutive
days. Nine aliquots of the 1/1 mixture of lecithin and sphingomyelin
were run each day. The mean L/S ratio was 0.96 (SD
0.20, CV 21.3%).
Clinical
Studies
We quantitated phospholipids in 22 specimens. Phosphatidylglycerol
was identified in all these, with L/S ratios ranging
from 2.9 to 15.3. None of these infants subsequently developed
the respiratory distress syndrome.
L/S ratios were assessed by our procedure and by the
method of Gluck et al. (10) in 30 paired specimens. There was
clustering of ratios by the latter method, in contrast to the
broader distribution by ours, which suggests that the present
method discriminates better (Figure 3). Two specimens obtained
at cesarean section gave low L/S ratios (1.8 and 1.9) by
the method of Gluck et al. (10); our method gave higher values
(3.0 and 2.7, respectively). Fluid from the former case also
contained phosphatidyiglycerol. Neither case developed the
respiratory distress syndrome.
Discussion
In this high-performance thin-layer chromatographic system,
silica gel is used as in conventional thin-layer chromatography,
but the particle size of the adsorbent material is
more uniform, about 7 sum. This allows for a densely packed
thin layer, which results in better resolution between similar
phospholipids (Figure 1). In conventional thin-layer chromatography,
in general, the particle distribution is in the range
of 5 to 30 zm; this less-dense adsorbent layer results in larger
and more diffuse spots.
The short-bed continuous-development chamber can better
404 CLINICAL CHEMISTRY, Vol. 26, No. 3, 1980

0
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A
Slope 0.20
1 2 3 4 5
Commercial Lecithin (mg)mixed with 1mg Sphingomyelin
Fig. 4. Sensitivity of the present method (HP-TLC) and that of
Gluck et al. (10) (Borer’s method) compared by use of commercially
prepared L/S mixtures
The slope for the present method is nearly three times greater, indicating its
greater sensitivity to changes in iecithin/sphingomyelin concentration
resolve substances with similar Rf’s. The Rf’s of substances
generally vary with solvent (mobile phase) strengths (8), but
if solvent concentrations are changed too much, the solvent
may traverse the chromatography plate before the substances
have moved from the origin. The short-bed continuous-development
chamber overcomes this difficulty because the
thin-layer chromatographic plate protrudes out of the otherwise
sealed chamber, permitting the solvent to move up the
plate and evaporate outside the chamber. Therefore, solvent
is continuously moving up the plate at a constant velocity. In
addition, as the distance to be traversed by the solvent is decreased,
solvent velocity increases (9). This continuous
movement of solvent results in resolution equivalent to that
with a much longer chromatographic plate or column, and the
high solvent velocity shortens the time required.
We found phosphatidylinositol and phosphatidylcholine
to have similar R1’s in solvent systems utilized by other investigators
(4). This was also true of phosphatidylsermne and
phosphatidylethanolamine. Thus, to achieve separation, we
used the short-bed continuous-development system and
added petroleum ether to reduce the polarity of the solvent.
The greater sensitivity of the present method as compared
with commonly used planimetric evaluation of L/S ratios (10)
is emphasized by comparison of the slopes obtained (Figure
4). The slope for the present method is nearly three times that
for the method of Gluck et al. (10), indicating that our system
gives a greater change in the calculated L/S ratio for a change
in the L/S weight mixtures. Although we did not obtain
identical values for commercial L/S mixtures and L/S ratios
determined by our procedure, there is a constant proportion
between the two (Figure 4), as there also is for the method of
Glucketal. (10).
The popularity of the method of Gluck et al. lies in avoiding
the need for sulfuric acid charring; our method also has this
advantage. In addition, densitometric quantification of
phospholipids enables a more rapid assessment of all phospholipid
bands than planimetry.
With our system, one-dimensional separation of the major
phospholipids currently being evaluated in predicting fetal
lung status is possible. Studies by Hallman et al. (3) have
suggested that phosphatidylethanolamine and phosphatidylserine
make up a significantly higher percentage of total
phospholipids in infants who develop the respiratory distress
syndrome. Isolation and quantification of these phospholipids
from amniotic fluids may prove to be an indicator of immature
fetal lung status, in contrast to phosphatidylglycerol, which,
when present, indicates mature fetal lung status (11, 12). The
converse, however, is not true: the absence of phosphatidylglycerol
does not necessarily indicate an immature lung.
Therefore, phosphatidylethanolamine and phosphatidylserine
may yield useful information in complicated pregnancies,
when the L/S ratio is frequently high despite immature lung
status (3).
References
I. Gluck, L., and Kulovich, M. V., Lecithin/sphingomyelin ratios in
amniotic fluid in normal and abnormal pregnancy. Am J. Obstet.
Gynecol. 115, 539-546 (1973).
2. Cunningham, M. D., Desai, N. S., Thompson, S. A., and Greene,
J. M., Amniotic fluid phosphatidylglycerol in diabetic pregnancies.
Am. J. Obstet. Gynecol. 131, 719-724 (1978).
3. Hallman, M., Feldman, B. H., Kirkpatrick, E., and Gluck, L.,
Absence of phosphatidylglycerol (PG) in respiratory distress syndrome
in the newborn. Pediat. Res. 11, 714-720 (1977).
4. Tsai, M. Y., and Marshall, J. G., Phosphatidylglycerol in 261
samples of amniotic fluid from normal and diabetic pregnancies, as
measured by one-dimensional thin-layer chromatography. Clin.
Chem. 25, 682-685 (1979).
5. Gotelli, G. R., Stanfill, R. E., Kabra, P. M., et al., Simultaneous
determination of phosphatidylglycerol and the lecithin/sphingomyelin
ratio in amniotic fluids. Clin. Chem. 24, 1144-1146 (1978).
6. Manual of Methods for Chemical Analysis of Water and Wastes
(EPA-625/6-74-003), U. S. Environmental Protection Agency,
Washington, D. C., 1974, pp 249-255.
7. Gluck, L., Kulovich, M. V., Borer, R. C., et al., Diagnosis of the
respiratory distress syndrome by amniocentesis. Am. J. Obstet. Gynecol.
109, 440-445 (1971).
8. Perry, J. A., A new look at solvent strength selectivity and continuous
development. J. Chromatogr. 165, 117-140 (1979).
9. Shortbed/Continuous Development Technical Manual, Regis
Chemical Co., Morton Grove, IL.
10. Gluck, L., Kulovich, M. V., and Borer, R. C., Estimates of fetal
lung maturity. Clin. Perinatol. 1, 125 (1974).
11. Kulovich, M. V., Hallman, M. B., and Gluck, L., The lung profile.
I. Normal pregnancy. Am. J. Obstet. Gynecol. 135, 57-63 (1979).
12. Kulovich, M. V., and Gluck, L., The lung profile. II. Complicated
pregnancy. Am. J. Obstet. Gynecol. 135, 64-70 (1979).
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