Volume 328. number 1.

2, 13-16 FEBS 12788 August 1993

0 1993 Federation of European Biochemical Societies 00145793/93/$6.00

Detection of complex gangliosides in human amniotic fluid

Ricardo Rueda”, Khalil Tabshb and Stephan Ladisch”,’

Departments of “Pediatrics and bObstetric.s. UCLA School of Medicine. Los Angeles, CA 90024, USA and ‘Center_for Carzcer und
Transplantation Biology Children’s Nutional Medical Center, 111 Michigan Avenue. NW: Washwzgtou. DC 20010, USA

Received 16 April 1993: revised version received 14 June 1993

Gangliosldes are possibly very potent immunosuppressive molecules. Here we show that human amniotic fluid contains high concentrations of
a number of previously unnoted, structurally complex and highly polar ganghosides These unusual molecules are present early m pregnancy (first
trimester), increase in concentration with gestational age, and reach maximum levels (0.8 PM) at term. Smce similar ganghosides have been detected
in human placenta, trophoblast. and amnion. we suggest that these molecules are shed into the amniotic fluid bathing these tissues.

Shed ganghoside, Amniotic fluid

1. INTRODUCTION quantitatively, ganglioside synthesis [17]. To our sur-

prise. we detected a series of structurally complex and
During pregnancy, there is an intense proliferation of highly polar gangliosides in human amniotic fluid as the
the haploidentical fetal allograft, which should result in major and almost exclusive components.
its rejection. Why this does not occur remains an
enigma. However, the maternal-fetal interface may
have a role in prevention of fetal rejection [l-6], shed- 2. MATERIALS AND METHODS
ding immunosuppressive molecules which can be found
in the amniotic fluid, which itself has immunosuppres- Aliquots of amniotic fluid samples which had been obtained from
sive properties [7710]. One such class of immunosup- pregnant women at 1640 weeks of gestation by transabdommal am-
niocentesis under sterile conditions were studied. The samples were
pressive molecules is the gangliosides, sialic acid-con-
cooled to 4°C immediately and rapidly processed.
taining glycosphingolipids which are embedded in cell After an mittal analysis of unfractionated ammotic fluid, all samples
membranes by their ceramide portion. were fractionated by centrtfugation according to the scheme shown m
The shedding of cell surface membrane components Fig. 1 Cells (mostly epithelial) present in the ammotic fluid were
such as gangliosides is characteristic of rapidly prolifer- removed by centrtfugation at 800 x g for 15 min at 4°C. and the
supernate was recentrifuged at 8,000 x g for 15min at 4°C. to remove
ating cells [ 111. Therefore, gangliosides from the mater- cell fragments In some samples, particularly those obtained at the end
nal-fetal system might be found in the amniotic fluid. of pregnancy. it was also possible to isolate surfactant as a floatmg
Since the shedding of tumor gangliosides inhibits nor- white layer after centrifugation. To determine the qualitative and
mal cellular immune responses [ 121. we hypothesize that quantitative distribution of ganghosides in human amniotic fluid and
its particulate fractions and m surfactant, each component was proc-
such shedding could likewise modify the immunological
essed separately under identical conditions
interaction between the fetus and the mother. The ob-
ject of this study therefore was to characterize the gan- 2 2 Gunglrosidr pmfcation
glioside content of human amniotic fluid. The total ganghostdes of the samples were purified by total hpid
We expected the ganglioside composition of human extraction, diisopropyl ether (DIPE)/l-butanol/aqueous phase parti-
tion and Sephadex G-50 gel filtration as developed for the isolation
amniotic fluid to reflect the ganglioside composition of ofgangliostdes from fluids [18,19]. Total lipid extracts were preparated
the placenta, trophoblast, and amnion, in which the usmg 2 ml chloroform/methanol (C:M) (1.1.v/v) per ml amniotic fluid
ganglioside G,, is predominant [13-l 61, because gangli- and 20 ml C/M (1: 1. v/v) per gram of cells. cell fragments or surfactant.
oside shedding generally reflects, qualitatively and All samples were extracted twice The extracts were pooled and clari-
fied by centrifugation (750 x g for 10 min at 4°C) to remove particu-
late matter, reduced to approximately l/4 of then original volume by
rotary evaporation, and cooled to -20°C. Additional msoluble mate-
Correspondence address. S. Ladtsch, Center for Cancer and Trans- rial was removed by centrifugation and the final total lipid extracts
plantation Biology, Children’s National Medical Center. 111Michi- were taken to dryness
gan Avenue. NW, Washington, DC 20010-2970. USA. Fax: (I) (202) Partitionmg was accomplished by dispersing the dried total lipid
745 5685. extracts in DIPE/l-butanol (60:40, v/v). with several minutes of
vortexing and bath somcation. 4 ml of the organic solvent mixture was
Abbrewztions. C, chloroform; M, methanol; HPTLC, high-perform- used for the total lipid extract of each 10 ml of amniotic fluid and 20
ance thin-layer chromatography; LBSA, lipid-bound sialic acid. ml for the total lipid extract of each gram of cells. cell fragments. or

Published by Elsevier Selence Publishers B K 13

Volume 328, number 1.1 FEBSLETTERS August 1993

surfactant Next. distilled Rater (for ammotic fluid) or 0.1% aqueous

Fresh Amniotic Fluid
NaCl (for cells, cell fragments and surfactant). one half of the volume
of the organic solvent, was added with vortexing and somcation. After
Centrifuge
centrifugation at 750 x g for 10 min to separate the two phases, the 8OOg x 15min
upper organic phase (contammg the neutral hptds and phospholipids)
was carefully removed. The ganglioside-contammg lower aqueous
phase was re-extracted twice with the original volume of fresh orgamc f-l
Pellet (PII Supernatant
solvent. and the final aqueous phase was lyophilized. The samples
(epithelial cells)
were redissolved m a small volume of distilled water. somcated. and
subjected to Sephadex G-50 gel filtration. usmg distilled water as the Centrifuge
S.OOOg x 15mln
elutmg solvent. Gangliosides were recovered m the void volume peak,
lyophthzed. redissolved m a small volume of C/M (I.1 1, and centri-

3 t +
I

fuged to remove insoluble restdual protems co-extracted with gangli-

osides The purified ganghosides were recovered from the clear super-
nate and stored under N2 at -20°C Pellet (Pp) Clarified Surfactant (S)
(cell fragments) Fluid (AF) (floating layer)

Ftg. 1. Separation of amniotic fluid fractions.

Ganghosides were quantttated as nmol hptd-bound siahc acid
(LBSA) by the modified calorimetric resorcmol assay [ZO]. High-per-
formance thm-layer chromatography (HPTLC) of gangliosides was
the detection of sphingosine by positive ion FAB mass
performed usmg 10 x 20 cm precoated Silica Gel-60 HPTLC plates (E.
Merck, Darmstadt. Germany) Two HPTLC development systems
spectrometry after acid hydrolysis [23] of the total gan-
were used to separate the glycophingohpids The first was the solvent glioside extract (not shown). Significantly, G,,, the
system widely used for ganghoside analysts, C/M/0.25% CaCl? 7HZ0 most prevalent extraneural ganglioside. was undetecta-
(60:40,9. v/v/v) [20] The second. described by Rosner [?l]. IS particu- ble in the amniotic fluid. This experiment gave the first
larly suitable for vtsuahzmg highly polar highly complex ganghosides.
suggestion that human amniotic Auid is characterized
In this method gangliosides are applied to the HPTLC plate which IS
then warmed for 1 h at 50°C. After cooling, the plates are developed by an easily visible concentration of highly complex
by consecutive runs (m the same direction and at 38°C) m two differ- gangliosides, and a paucity of the usually prevalent ex-
ent solvent systems: (a) C/M/l2 mM MgClJl5 N NH,OH (60,37:7,3. traneural ganglioside, GSI.
v/v/v/v). and (b) C/M/l2 mM MgCl: (58:-10:9. v/c/v). Ganghostdes
here vtsuahzed as purple bands with resorcmaol reagent [22]
3.2. Changes in i~~~mmarnnioticjuid ganglioside content
btsitfigestationul uge
3. RESULTS
Seventeen human amniotic samples, freshly obtained
3.1. Hunmn amniotic fluid gungliosides and immediately clarified by centrifugation, were stud-
In initial experiments we analyzed the total gangli- ied to determine the range of ganglioside concentration
osides of four samples of unseparated amniotic fluid in human amniotic fluid. and the effect of gestational
obtained at different periods of gestation, using the age. Table I shows that the ganglioside concentration
standard ganglioside HPTLC solvent system of C/M/ in amniotic fluid increases at least fourfold from the first
0.25% CaCl,.H20 (60:40:9, v/v/v) (Fig. 2). Only trimester of pregnancy until term. with the median con-
traces (< 50 pmol LBSA/ml amniotic fluid) or no re- centration rising from 0.2 ,DM LBSA at 16 weeks to 0.8
sorcinol- positive (ganglioside) bands were seen in the PM LBSA at the end of pregnancy.
region of migration of most of human brain gangli- To determine whether the total ganglioside composi-
osides in this solvent system (i.e. between G,, and tion undergoes qualitative change with increasing gesta-
G,,,). However, a very densely staining resorcinol-pos- tional age. the total gangliosides isolated from amniotic
itive band is visible near the origin of the HPTLC in
each of the four amniotic fluid samples (Fig. 2). This
band has an exceedingly slight but consistent migration --GM3
above the origin of the HPTLC. suggesting that these : \,
molecules may be very complex gangliosides. To inves-
tigate these molecules further, we therefore chose a sol- ;”

vent system which is particularly suitable for resolving GDla-

slowing migrating gangliosides [21]. r*&k
In the first experiment in which we applied this GQlb- Oscx
__.
method to a sample of pooled amniotic fluid, we de-
tected a series of four highly complex gangliosides HBG 16 17 28 36 HPG
which were the main components of this fluid (Fig. 3).
AF, Weeks Gestation
For comparison, total human brain and plasma gangli-
osides are shown, demonstrating the strikingly slower Fig. 2. Ammotic fluid ganghostdes. Total purified ammotic fluid gan-
ghosides (AF) were separated by HPTLC usmg the solvent system,
migration of the human amniotic fluid gangliosides. all C/M/0.15% CaClz.2HZ0 (60.40.9, v/v/v). 2-5 nmol were spotted per
more polar than G,,,. The gangliosidic nature of these lane. HBG. human bram standard gangliostdes; HPG. human plasma
resorcinol-positive molecules was further supported by ganghosides. All bands were resorcmol-posttive.

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Volume 328, number 1,2 FEBS LETTERS August 1993

Only traces of the complex gangliosides predominant in

-GM3 the clarified fluid were detected in the cell pellets and the
iY
surfactant. Therefore, the major complex gangliosides
G Ml-- of amniotic fluid are not related to those of surfactant
GD1ci- or of epithelial cells found in the fluid.

G Tlb- -
4. DISCUSSION

Tissue gangliosides of the fetal-maternal system, in-

cluding of placenta [13-l 51, placental syncytiotropho-
HBG AF HPG blast [24] and meconium [25,26], have been character-
Ftg. 3. HPTLC separatton of highly polar amniotic fluid gangliosides. ized. Several glycoconjugates have been identified in
4 nmol total human amniotic fluid gangliosides were separated by amniotic fluid (e.g. lactosylceramide [27], blood group
development of the plate in C/M/l2 mM MgClJlS N NH,.H:O
I and i antigens [28] and galactosyloligosaccharides
(60:37:7:3, v/v/v/v), followed by C/M/l2 mM MgCl, (58:40,9, v/v/v).
Abbreviations as m Fig. 2 [29]), but little is known about the gangliosides, which
are difficult to purify when present only at low concen-
trations. The major finding of this study was that the
fluid obtained at different periods of pregnancy were major gangliosides of human amniotic fluid are a series
analyzed by the HPTLC method of Rosner [21]. As of highly polar and complex molecules. This is surpris-
shown in Fig. 4, over the period of gestation studied ing because the major ganglioside of placenta is G,,
(1640 weeks of pregnancy), the pattern of amniotic [13-l 51 and no other normal human tissue or fluid is
fluid gangliosides is constant. Furthermore, there are known to have these highly complex gangliosides as
not more than traces of simple ganglioside such as G,? major components.
in amniotic fluid. The striking constancy of individual The origin of complex gangliosides in human amni-
species from the first sample (16 weeks) to the final otic fluid is unknown. Amniotic fluid cells (epithelial
sample (end of pregnancy) suggest that the production cells) are excluded as a likely source by our studies
of these highly complex gangliosides continues. without because the major ganglioside of these cells is GM3 [30],
qualitative change, throughout pregnancy. and the complex gangliosides found in amniotic fluid
were absent in the cell fraction. The same is true of
3.3. Gangliosides of amniotic fluid cellular components surfactant. Human placenta does contain trace amounts
and surfactant of several highly polar neolacto series gangliosides
The above studies detecting complex gangliosides [15.31,32]. migrating near the origin by standard
were performed on amniotic fluid which had been clar- HPTLC. Syncytiotrophoblast and amnion also contain
ified by centrifugation to assure that gangliosides iso- such complex molecules [ 16,24,33]. Therefore, these tis-
lated were not associated with cellular or other particu- sues could be the origin of the highly complex gangli-
late matter contained in amniotic fluid. Removed from osides in amniotic fluid. By the process of shedding,
the amniotic fluid in this process are centrifugation pel- these molecules could accumulate and concentrate in
lets (which include epithelial cells and cell fragments) amniotic fluid during gestation.
and sometimes a floating lower density fraction, proba- What is the potential significance of the finding of
bly surfactant, especially visible in the case of samples
obtained at term. Gangliosides from these two minor
fractions. comprised at most only a few percent of the
-GM3
total amniotic fluid gangliosides. However, these frac-
tions yielded a surprising pattern (Fig. 5); in contrast to
the amniotic fluid gangliosides (complex), the predomi-
-GDZ
nant ganglioside in each of these fractions was G,,.

Table I
-GTlb
Ganghostde concentration of amniotic fluid: Effect of gestational age

Gestattonal Age Gangliosides @M)

(weeks)
Mean Range HBG ,I6 27 28 36, HPG

1&17(n=4) 0.2 0.1-0.3 AF, Weeks Gestation

27-29 (n = 4) 0.5 < 0.1~1.0 Fig. 4. Effect of gestational age upon human amniotic fluid gangli-
3435 (n = 2) 0.4 0.3. 0.4 ostde Samples obtamed in each trtmester were purtfied and compared.
3640 (?I = 7) 0.8 0.4-l .3 Abbrevtations as in Fig. 2. Solvent system: as m Fig. 3.

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Volume 328, number 1.2 FEBS LETTERS August 1993

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[I41 Portoukalian. J and Zwmgelstein. G. (1981) Int. J TISS. Reac
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