CLIN. CHEM. 27/3, 486-490
(1981)
Clinical Laboratory Determination of Phosphatidylglycerol: One- and TwoDimensional Chromatography Compared
Thomas L. Gross, Margaret V. Wilson, Paul M. Kuhnert, and Robert J. Sokol
in
lipids in AF, if reliable, might be faster and less expensive and
amniotic fluid, including phosphatidylglycerol
and phosphatidylinositol,
in addition to lecithin and sphingomyelin
improves prediction of neonatal pulmonary function. In this
study, we evaluated a two-dimensional technique for
separating and measuring these phospholipids and compared it with a simpler one-dimensional
procedure. The
two-dimensional
technique
was adapted to readily available commercial plates, and a preheating step was introduced to avoid shattering of the plates during charring. The
R values, reproducibility
of each technique, and the correlation between them were examined. Even though the
one-dimensional
technique is faster and less expensive,
we recommend the two-dimensional
method for clinical
use because of better precision (CV for phosphatidylglycerol 15% vs 21 %) and clearer results when relatively
little phosphatidylglycerol
is present. The one-dimensional
allow several samples to be separated on a single plate.
Earlier investigators described one-dimensional thin-layer
chromatographic
techniques for separating phospholipids in
animal tissues (8). Others used a one-dimensional procedure
in which two different
solvent systems were used successively
(9). Other, more recent one-dimensional
techniques have been
adapted to use in separating phospholipids
in AF, but thus
Reportedly,
procedure
determination
is unreliable
of several
when
blood
phospholipids
or meconium are
present. In addition, interfering compounds co-migrate with
phosphatidylglycerol,
phosphatidylethanolamine,
and
phosphatidylserine
in the one-dimensional
technique.
Before any one-dimensional
lipid separation is adopted for
clinical use, it should be critically compared to the twodimensional procedure.
AddItIonal Keyphrases:
fetal status
fluid . respiratory distress syndrome
.
US ratio
amniotic
phospholipids
Over the years, various methods have been proposed for
assessing fetal lung maturity
by analyzing amniotic
fluid
(AF).1 One of these, evaluation of the lecithin/sphingomyelin
(L/S) ratio, is widely used as the clinical test for predicting the
respiratory
distress syndrome (1). A “mature”
L/S ratio appears to be very reliable in predicting fetal lung maturity, but
false-positive results do occur (2). The L/S ratio has been less
reliable in predicting
fetal lung immaturity
(2, 3). Recent
studies (4) suggest that additional
information
concerning
fetal pulmonary maturity and gestational age can be obtained
by measuring two other AF phospholipids,
phosphatidylglycerol and phosphatidylinositol.
Methods have been developed for separating the various
phospholipids
in AF-lecithin
(L), sphingomyelin
(S),
phosphatidylglycerol
(PG), phosphatidylinositol
(P1), phosphatidylserine
(PS), and phosphatidylethanolamine
(PE).
The two-dimensional
thin-layer chromatographic
technique
of Gray (5) for separating
acidic phospholipids
was first
adapted to AF lipids by Hallman et al. (6), and is now widely
used (2, 7). However, a one-dimensional
thin-layer
chromatographic method for separating major and minor phosphoDepartment of Obstetrics and Gynecology and the Perinat.al
Clinical Research Center, Cleveland Metropolitan General Hospital/Case Western Reserve University, 3395 Scranton Rd., Cleveland,
OH 44109.
1 Nonstandard
abbreviations used: AF, amniotic fluid; L, acetone-precipitable lecithin; S, sphingomyelin; PG, phosphatidylglycerol; P1, phosphatidylinositol;
PS, phosphatidylserine; and PE,
phosphatidylethanolamine.
Received Oct. 6, 1980; accepted Jan. 2, 1981.
486
CLINICAL CHEMISTRY, Vol. 27, No. 3, 1981
far none of them gives complete resolution of PS and P1 or PS
and PE (10-12).
Our purpose in this study was to determine which of two
techniques for separating AF phospholipids
would be the more
appropriate
for routine
use in the clinical laboratory, the
technique
of Haliman
et al. (6) or a one-
two-dimensional
dimensional
procedure similar to that reported by Tsai and
Marshall (11). We slightly modified the technique of Hailman
et al. (6) so that commercially
available
thin-layer
chromatographic
plates could be used.
Materials and Methods
Samples:
We studied
AF samples
from 59 patients
who
required amniocentesis.
Of these, 32 had previously had Cesarean sections, 10 had diabetes mellitus, six were Rh negative
and sensitized, six had pre-eclampsia,
and five had chronic
hypertension.
The mean gestational
age was 39 (SD 2.2)
weeks. We assayed a single sample from each patient.
In studying the correlation between these two techniques,
we used amniotic fluid that was uncontaminated
with blood
or meconium, but we also studied the effect of such contamination. The fluids contaminated with blood contained no more
than 3 mL of blood per deciliter.
Method: Fluids were centrifuged at 1000 X g for 5 mm and
the supernatant
fluid was analyzed within 2 h or stored frozen
until analysis. Phospholipids
were extracted by use of the
method of Gluck et al. (1). After extraction, the lipid residue
was dissolved in 20 iiL of chloroform.
The sample was then
divided and analyzed by both the one- and the two-dimensional techniques. The volume of lipid extract applied to the
plate ranged from 5 to 10 sL, depending on a visual estimate
of the amount of dried lipid present after the precipitation
with acetone. Commercially
available thin-layer
chromatographic plates, prepared with 50 g/L ammonium
sulfate, 20
X 20cm, with a 200-sm coating of silica gel H (Analtech, Inc.,
Newark, DE 19711) were used for both techniques. We chose
these plates because they are made to the specifications of the
plates in the widely used two-dimensional
procedure of Gluck
et al. (personal communication).
Thin-layer
chromatography:
For the two-dimensional
separation, we altered the solvent system described by Gluck
et al. (personal communication)
slightly, because it did not
give a clear separation with the commercial plates. The plates
were first developed for 10 cm with a mixture of chloroform,
methanol,
water, and 17.4 mol/L acetic acid (162.5/65.0/
10.0/0.5 by vol). The plate was then turned 90#{176}
so that the
original right side was the origin and a second solvent system
(tetrahydrofuran,
methylal, methanol,
and 2 mol/L ammonium hydroxide, 120/80/35/10 by vol) was allowed to ascend
10cm. The plates were activated by heating at 110 #{176}C
for 30
mm before the first migration and were dried at 70 #{176}C
for 5
Table 1. R Values for Phospholipids in 10
Different Amniotic Fluid Samples with the One-
and Two-Dimensional Techniques
On.-dlm.nsioeal
R values (X 100) a
Two-dlm.nslonAl
1st directIon
2nd dlr.ctlon
12±2
Does notmlgrate
22±2
Doesnotmigrate
Sphlngomyelin
LecIthin
12±2
22±2
P1
PE
32±2
44±3
33±2
48±2
PS
PG
46 ± 3
65 ± 3
50 ± 3
62 ± 3
24±2
12 ± 1
Does not migrate
52 ± 4
1Mean ± standarddeviatIon.
min before the second solvent system was used. Before each
migration step, the plates were allowed to cool until they were
just warm to the touch.
In the one-dimensional technique, the solvent system used
was the same as the first in the two-dimensional procedure,
and the solvent was allowed to ascend 12 cm from the origin.
After development, the plates from both the one- and two-
dimensional procedures were preheated in an oven set at 110
#{176}C,
then charred on a hot plate at 280 #{176}C
for 10 mm to make
the compounds
visible. Initially, we used standards to assure
proper identification: synthetic dipalmitoyl lecithin, bovine
brain sphingomyelin
and phosphatidylserine,
egg-yolk
phosphatidylglycerol
and phosphatidylethanolammne,
and
soybean phosphatidylinositol
(all from Sigma Chemical Co.,
St. Louis MO 63173). The concentration of all standards used
was 1 g/L.
To obtain semi-quantitative results for each phoepholipid,
the L, S, PG, P1, PE, and PS spots were scanned with a
transmission
densitometer
in dual-beam mode with attached
linear recorder and integrator (Kontes Co., Vineland, NJ,
08360). The figures for the densitometric scans of L, PG, P1,
PE, and PS were totaled and each compound was expressed
as a percentage of the total. On the one-dimension plate, the
combined peak of PE and PS was expressed as a single
value.
The one- and two-dimensional plates from each AF sample
were first compared visually. The correlation between the two
techniques was evaluated quantitatively
by comparing the
results for each compound in the 59 amniotic fluid samples.
Finally, the precision of both techniques was assessed by
calculating the coefficients of variation for each compound,
based on 10 replicate determinations of one AF sample.
Fig. 1. A two-dimensional
chromatogram of AF from a patient
at 40 weeks’ gestation
L, theacetone-preclpltablofractionof lecithin. SOIL, theacetone-soluble
fraction
of lecithin.
The unknown (Unk) has been Identif
led (5) as lysoblsphosphatldic
acid.For otherabbreviations
used Inthese figures,
see footnote’. Some of the
patterns
shown In these figures
have been cropped slightly
compounds that migrated
near the PG spot in the tmi-dimension system, as shown in AF no.2 (Figure 3A and C). (d)
On one occasion a non-bloody AF appeared to have PG on the
one-dimensional plate, when there was clearly none in the
two-dimensional (Figure 4A and B). Both of these last two
circumstances could obscure the detection of and possibly
cause a false-positive for PG.
The ease of detecting the phospholipid spots visually and
Results
Separation
of phospholipids.
nique gave complete resolution
The two-dimensional
tech-
of L, S, PG, P1, PE, and PS.
The one-dimensional technique gave complete separation of
L, S, PG, and P1, but not of PE and PS. The Rf values (mean
±SD for 10 samples) for each phospholipid analyzed were
reproducible with both techniques (Table 1).
In visually and quantitatively
comparing the one- and
two-dimensional procedures, we found that the latter technique gave clear resolution of the phospholipids in all fluids
analyzed.
We noted interfering
compounds
in the one-dimensional technique in the following four circumstances:
(a)
All fluids were found to contain a compound that co-chromatographed
with PE and PS in the one-dimensional
tech..
nique (Figures 1 and 2). (b) In the one-dimensional system,
meconium was found to contain various compounds that cochromatographed
with either L, S, P1, or PE, and often totally
obscured the L/S ratio, as illustrated by AF no.1 in Figure 3A
and B). (c) In some cases, blood-contaminated
AF contained
Fig. 2. A one-dimensional clrwomatogram of two AF specimens:
one with PG (1), one wIthout (2)
The PE/PS spotcontains
a third
component shown as an unknown (Link) In Figure
1. PGand P1 standardsare also shown
CLINICAL CHEMISTRY,
Vol. 27, No. 3, 1981
487
A
$
B
4
Fig. 4A. One-dimensional chromatogram of AF from a patient
with acute hydramnios
IIsat 24weeksof gestation,
2at 25 weeks.BothappeartohaveatraceofPG
Unk
PL
=
PS
present
B. Two-dimensional
PG to be present
chromatogram of fluid 2 in A, showing no
P1.
C
Fig. 3A. One-dimensional chromatogram of a meconlum-stained
AF (1) and an AF stained with maternal blood (2). B. Two-di-
mensional chromatogram of the same meconium stained fluid
(1) shown In A
Meconlum obscuresthe L and S In the one-dimensional
patternsbut not the
two-dImensional
pattern
C. Two-dimensional
chromatogram of the same blood-stained
fluid(2) shown in A
The bloodcontaminatedfluid appears to have PG In the one-dimensional
pattern
butnone In the two.dlmenslonal
pattern
488
CLINICAL
CHEMISTRY,Vol.
27,
No. 3,
1981
quantitating them with a densitometer varied with the two
techniques. With the two-dimensional method, the phospholipid spots were easier to detect and quantitate;
with the
one-dimensional technique the PG spot was diffuse, and
therefore difficult to quantitate.
Precision. The precision for each technique was evaluated
by determining the reproducibility of both procedures. Table
2 shows the mean, standard deviation,
and coefficient
of
variation for 10 replicate determinations.
The standard deviation and coefficient of variation of PG was greater with the
one-dimensional
technique than with the two-dimensional
system. The other compounds either varied similarly
(L/S,
L, and P1) or were impossible to compare (PE and PS).
Correlation of two techniques.
Correlation of the individual
components as determined by the two techniques yielded the
Table 2. LIS Ratio and the Individual Phospholipid
Components
in Percentage
of Total Phospholipid
in Amniotic Fluid a
On.-dlm.nelonal
procedure
X(SD)
L/S
Lecithin
PG
P1
PE
PS
Two-dImensIonal
-
CV,
separation that this compound is separated from PE and PS
only on the second migration.
There are additional problems with the one-dimensional
technique. The ammonium sulfate in the thin-layer chromatographic plates inhibited charring and this may lead to errors
when only a small amount of PG is present. Others (11) have
noted this as well, and have sprayed the plates with sulfuric
acid to improve charring, but this increases the potential for
uneven charring due to non-uniform spray.
Although the coefficients of variation for the individual
phospholipid components were generally very similar for the
one- and two-dimensional technique, the reproducibility for
PG appeared to be better in the two-dimensional technique.
This may be related to the diffuseness of the PG spot and the
difficulty in detecting small amounts of PG in the one-di-
proc.dure
_________________
%
-#{149} X (SD)
CV, %
2.05 (0.21)
10
58.70 (3.65)
6.20 (1.32)
20.50 (2.22)
14.60(3.92)”
6
21
11
27
2.28 (0.15)
63.50
7.00
12.25
6.40
10.80
(4.41)
(1.07)
(1.67)
(1.35)
(2.10)
7
7
15
14
21
19
a Databasedon 10 replIcatedeterminations
of the same AF specimen In oneand two-dimensIons.
b Representscombined spot of P6 and PS.
following r values for the 59 AF samples: L/S = 0.88, L% =
0.41, PG% = 0.84, P1% = 0.86 (all p <0.01), and (PE + PS)%
= 0.35, p <0.05. In the one-dimensional method the combined
PE and PS spot actually contains a third, unknown component (Figures 1 and 2). The correlation was weakest when the
densitometric scan of the combined PE, PS, and unknown
spot on the one-dimensional plates was compared with the
sum of the densitometric scans of the individual PE and PS
spots on the two-dimensional plates.
Assay time was 90 min for the one-dimensional method, 150
mm for the two-dimensional technique.
Discussion
The two-dimensional
procedure reliably separated all
phospholipid components, as previously described (6). Although the one-dimensional procedure adequately separated
the components most important
clinically (L/S, PG, and P1),
it presented several problems. We conclude that the twodimensional technique is the better and more appropriate
technique for use in the clinical laboratory, because of potential interference by other compounds co-chromatographing
with the important phospholipids in the single-dimensional
procedure. Others (11) have reported that, in this technique,
blood and meconium can obscure the L and S spots, a finding
we confirm. Moreover, meconium may also obscure the PT,PE,
or PS spots, and blood-stained AF on occasion contains
compounds that can obscure PG or cause a falsely positive PG
spot. The latter finding is not in agreement with a previous
report of a single-dimension technique (11). Finally, on one
occasion an immature, non-bloody AF appeared to contain
PG on the one-dimensional plate, but PG was clearly absent
in the two-dimensional. Interference from compounds other
than the phospholipids of interest could lead to inappropriate
clinical decisions in diagnosing fetal maturity, and this may
explain a finding reported by Cunningham et al. (13). These
investigators combined chromatography on a diethylaminocellulose column with one-dimensional thin-layer chromatography to separate AF phospholipids and reported abovenormal PG in AF from normal and diabetic pregnancies of less
than 33 weeks’ gestation. Their one-dimension thin-layer
chromatographic step appears similar to ours and the falsepositive PG spot we occasionally saw with the one-dimension
technique could account for the apparently above-normal PG
that they observed in premature patients. Finally, the combined PE and PS spot described in a one-dimensional procedure by Tsai and Marshall
(11) actually contains
another
interfering
compound,
previously
identified
by Gray (5) as
lysobisphosphatidic
acid. It is apparent
when the one-dimensional plate pattern is compared with the two-dimensional
mensiotlal system.
Although the one-dimensional method is faster and more
economical, we believe the two-dimensional technique of
Hallman et at. (6) is the better choice for implementation in
the clinical laboratory because it is more unequivocal.
Several important technical considerations need mention.
Commercially available thin-layer chromatographic plates
can be used, but the solvent ratios modified as described are
required for good separation. Also after activation, the plates
should be allowed to cool until they are just warm to the touch,
before they are placed in the first tank, otherwise lecithin and
sphingomyelin and PT and PE are poorly separated. Others
(10) have found commercial thin-layer chromatographic
plates unacceptable for high-temperature charring because
of a tendency to shatter. We avoid this by pre-heating the
plates to 110 #{176}C
in an oven prior to placing them on the 280
#{176}C
hot plate.
Supported in Part by USPHS Grants 5M01-RR00210
Division of Research Resources.
and IP5OHD,
References
1. Gluck, L., Kulovich, M. V., Borer, R. C., et at., Diagnosis of the
respiratory distress syndrome by amniocentesis. Am. J. Obstet. Gynecol. 109, 440-445 (1971).
2. Kulovich, M. V., Hailman, M. B., and Gluck, L., The lung profile
I. Normal pregnancy. Am. J. Obstet. Gynecol. 135, 57-63 (1979).
3. Nakamura,
J., Roux, J. F., Brown, E. G., and Sweet, A. Y., Total
lipids and the lecithin-sphingomyelin
ratio of amniotic fluid: An antenatal test of lung immaturity. Am. J. Obstet.Gynecol. 113,363-366
(1972).
4. Kulovich, M. V., and Gluck, L., The lung profile II. Complicated
pregnancy. Am. J. Obstet. Gynecol. 135, 64-70 (1979).
5. Gray, G. M., Chromatography
of lipids. II. The quantitative isolation of the minor (acidic) phospholipids and of phosphatidylethanolamine from the lipid extracts of mammalian tissues. Biochim.
Biophys. Acta 137, 519-524 (1967).
6. Hallman, M., Kulovich, M. V., Kirkpatrick, E., et al., Phosphatidylinositol and phosphatidylglycerol
in amniotic fluid: Indices of lung
maturity. Am. J. Obstet. Gynecol. 125,613-617 (1976).
7. Golde, S. H., and Mosley, G. H., A blind comparison study of the
lung phospholipid profile, fluorescence microviscosimetry,
and the
lecithin/sphingomyelin
ratio. Am. J. Obstet. Gynecol. 136, 222-227
(1980).
8. Parker, F., and Peterson,
N. F., Quantitative
analysis of phospholipids and phospholipid
fatty acids from silica gel thin-layer
chromatograms. J. Lipid Res. 6,455-460(1965).
9. Skipski, V. P., Barclay, M., Reichman, E. S., and Good, J. J., Separation of acidic phospholipids by one-dimensional
thin-layer chromatography. Biochim. Biophys. Acta 137, 80-89 (1967).
10. Gotelli, G. R., Stanfill, R. E., Kabra, P. M., et al., Simultaneous
determination of phosphatidyiglycerol
and the lecithin/sphingomyelin
ratio in amniotic fluid. Clin. Chem. 24, 1144-1146 (1978).
11. Tsai, M. Y., and Marshall, J. G., Phosphatidylglycerol
in 261
samples of amniotic fluid from normal and diabetic pregnancies as
CLINICAL CHEMISTRY, Vol. 27, No. 3, 1981
489
measured by one-dimensional
thin-layer chromatography.
Clin.
Chem. 25,682-685 (1979).
12. Kolins, M. D., F.p8tein, E., Civin, W. H., and Weiner, S., Amniotic
fluid phoepholipids measured by continuous-development
thin-layer
chromatography.
Clin. Chem. 26,403-405(1980).
13. Cunningham, M. D., Desai, N. S., Thompson, S. A., and Greene,
CLIN.CHEM. 27/3,
J. M., Amniotic fluid phosphatidylglycerol
in diabetic pregnancies.
Am. J.Obstet.Gynecol.131,719-724 (1978).
14. Mitnik, M. A., DeMarco, B., and Gibbons, J. M., Amniotic fluid
phosphatidylglycerol
and phosphatidylinositol
separated by stepwise-development thin-layer chromatography. Clin. Chern. 26,
277-281 (1980).
490-492(1981)
Low Results for Inorganic Phosphorus with the SMACContinuous-Flow
Analyzer
E. Arthur Robertson, Ronald J. Elm,’ and Ernestine Johnson
Serum inorganic phosphorus concentrations
as measured
with the SMAC are lower than those found with other
methods. To resolve this problem we analyzed patients’
specimens and performed analytical recovery studies with
four different systems (SMAC, AutoAnalyzer
II, aca, and the
Fiske-SubbaRow
method). With the SMAC, results for
patients’ specimens are significantly lower (p <0.0001)
than with any of the other three methods. The SMAC recovered only about 87% of the added inorganic phos-
phorus. The value assigned to SMAC Reference I for inorganic phosphorus was 0.876 of the value obtained when
was analyzed by the reference method. Thus
there is a significant systematic error in the SMAC method
for inorganic phosphorusdetermination, attributable to an
the material
erroneous inorganic phosphorus concentration assigned
the SMAC calibration material by the supplier (Technicon).
Additional
error
#{149}
Keyphrases:
calibrator materials
reference interval
#{149}
analytical
When our laboratory determined the reference interval for
SMAC (Technicon Instruments
Corp., Tarrytown,
NY 10591)
inorganic phosphorus, it was low as compared with reference
intervals in standard clinical chemistry textbooks. Furthermore, the results from the survey programs of the College of
American Pathologists (CAP) showed that the mean result
for inorganic phosphorus as determined with SMAC5 was
significantly lower than that for most other analytical systems.
In an attempt to resolve this apparent discrepancy, we analyzed patients’ specimens, using four different
analytical
systems (SMAC, Technicon AutoAnalyzer, ace, and manual
Fiske-SubbaRow), and also performed analytical recovery
studies.
Clinical Chemistry Service, Clinical Pathology Department, Clinical
Center, National Institutes of Health, Bethesda, MD 20205.
Address correspondence
to this author at Building
220, NIH, Bethesda, MD 20205.
Received
490
Nov. 7, 1980; accepted
CLINICAL CHEMISTRY,
Dec. 5, 1980.
Vol. 27, No. 3, 1981
10, Room 5N-
Materials and Methods
Analytical
systems.
The classical manual Fiske-SubbaRow
method was performed, in which phosphate reacts with ammonium molybdate to form ammonium phosphomolybdate,
which is reduced with aminonaphthol sulfonic acid to produce
a blue color (1). Absorbance is measured spectrophotometrically at 650 nm. The method used with the AutoAnalyzer is
the reference method that Technicon recommends, and they
use it in assigning values to their SMAC calibration material
(2,3). It is similar to the Fiske-SubbaRow method, except that
stannous chloride is used as the reducing agent and the colored
complex is determined at 660 nm (2). With the Automatic
Clinical Analyzer (ace; Dupont Co., Instrument Products
Division, Wilmington,
DE 19898) the phosphorus method
involves the use of p-methylaminophenol sulfate and bisulfite
to reduce the phosphomolybdate
complex, which is then
measured at 340 nm. The phosphorus method used in the
SMAC differs significantly
in that the unreduced
phosphomolybdate complex, which also absorbs ultraviolet light, is
determined at 340 nm (4).
Calibration
materials
and standards.
The Fiske-SubbaRow and AutoAnalyzer systems were standardized for
phosphorus with National Bureau of Standards monobasic
potassium phosphate (SRM-186-Ic KH2PO4). We calibrated
the SMAC with SMAC Reference I (Technicon product no.
T03-7120) and the ace with DuPont calibration
materials
(lots E0613A and J0669A).
Study
design
and
statistical
analyses.
To compare
the
results obtained on clinical samples, we analyzed 25 patients’
sera on the SMAC and in triplicate by both the AutoAnalyzer
and the Fiske-SubbaRow methods. Results from the SMAC
were also compared with those from the aca for sera from
blood samples obtained from 354 NIH employees after a short
fast. The paired-sample Student’s t-test was used to compare
SMAC results to those of the other methods.
Recovery studies were done by adding 0.2 mL of aqueous
KH2PO4 (NBS SRM 186-Ic) or water to 1.8 mL of pooled
serum to obtain a series of solutions with known differences
in phosphorus concentration. These six solutions, covering
a range of 80 mgfL, were analyzed in triplicate by each of three