Transport mechanisms for long-chain polyunsaturated fatty acids
in the human placenta1–3
Asim K Dutta-Roy
KEY WORDS
Human placenta, placental membrane fatty
acid binding protein, p-FABPpm, long-chain polyunsaturated
fatty acids, fetal development, fatty acid uptake, BeWo cells
INTRODUCTION
Dietary essential fatty acids (EFAs) are essential for cellular
growth and metabolism, and membrane structure and function
(1–3). EFAs and their long-chain polyunsaturated fatty acid
(LCPUFA) derivatives are of critical importance in fetal and
infant development (4, 5). Because of the fundamental roles of
EFAs and LCPUFAs as structural elements and functional modulators (1, 2), it has been hypothesized that maternal, fetal, and
neonatal LCPUFA status are important determinants of health
and disease in infancy and later in life. The deposition of these
LCPUFAs in the fetus is rapid during growth, and it has been
suggested that inadequacy of LCPUFAs in critical membrane
lipids may lead to a failure to accomplish a specific component
of brain growth and irrevocable damage (4). Both the brain and
the retina are rich in the LCPUFAs arachidonic acid (AA;
20:4n26) and docosahexaenoic acid (DHA; 22:6n23), and a
sufficient supply of these fatty acids during pregnancy and the
neonatal period is critical to proper function (4, 5). The fetal
brain acquires <21 g DHA/wk during the last trimester of pregnancy (6). Evidence from various studies suggests that retinal
function and learning ability may be permanently impaired if
there is a reduction in the accumulation of sufficient DHA during intrauterine life (4, 5).
Overall maternal LCPUFA status declines steadily during
pregnancy (7). Maternal DHA status was found to be significantly lower in multiparas than in primiparas, suggesting that in
usual dietary conditions, pregnancy is associated with mobilization of DHA from a store that may not be readily replenished
after delivery (7). Preterm infants have significantly lower EFA
and LCPUFA statuses than do full-term neonates (8, 9). In fact,
the potential for dietary EFA deficiency has become a significant
issue for the nutrition of preterm infants, because they do not
receive the third-trimester intrauterine supply of DHA and AA.
The fatty acid pattern found in the walls of umbilical blood vessels indicates that the EFA status of a developing fetus is marginal
or barely sufficient (10, 11). Supplementation of pregnant mothers with LCPUFAs has been shown to improve neonatal LCPUFA
status (12). However, a better understanding of the biochemical
processes involved in fetoplacental LCPUFA metabolism and
transport is required before the optimum and safe levels of supplementation during pregnancy can be determined. This is particularly important for premature infants because they have low
blood DHA concentrations, which affect their eye and brain functions as measured by electroretinogram, cortical visual evoked
potential, and behavioral testing of visual acuity (9, 12, 13).
Numerous studies have shown that LCPUFA concentrations
are higher in fetal than in maternal circulation (4, 10, 11, 14), but
the underlying biochemical mechanisms controlling this phenomenon are not understood. To examine the role of the placenta in
the preferential accumulation of LCPUFAs in fetuses, we studied
1
From the Rowett Research Institute, Aberdeen, Scotland, United Kingdom.
Supported by the Scottish Executive Rural Affairs Department.
3
Address reprint requests to AK Dutta-Roy, Rowett Research Institute,
Aberdeen AB21 9SB, Scotland, United Kingdom. E-mail: adr@rri.sari.ac.uk.
2
Am J Clin Nutr 2000;71(suppl):315S–22S. Printed in USA. © 2000 American Society for Clinical Nutrition
315S
Downloaded from www.ajcn.org at Aker Sykehus Medisinsk Bibliotek on April 24, 2007
ABSTRACT
To understand the placental role in the processes
responsible for the preferential accumulation of maternal longchain polyunsaturated fatty acids (LCPUFAs) in the fetus, we
investigated fatty acid uptake and metabolism in the human placenta. A preference for LCPUFAs over nonessential fatty acids
has been observed in isolated human placental membranes as
well as in BeWo cells, a human placental choriocarcinoma cell
line. A placental plasma membrane fatty acid binding protein (pFABPpm) with a molecular mass of <40 kDa was identified. The
purified p-FABPpm preferentially bound with essential fatty acids
(EFAs) and LCPUFAs over nonessential fatty acids. Oleic acid
was taken up least and docosahexaenoic acid (DHA) most by
BeWo cells, whereas no such discrimination was observed in
HepG2 liver cells. Studies on the distribution of radiolabeled fatty
acids in the cellular lipids of BeWo cells showed that DHA is
incorporated mainly into the triacylglycerol fraction, followed by
the phospholipid fraction; the reverse is true for arachidonic acid
(AA). The greater cellular uptake of DHA and its preferential
incorporation into the triacylglycerol fraction suggests that both
uptake and transport modes of DHA by the placenta to the fetus
are different from those of AA. p-FABPpm antiserum preferentially decreased the uptake of LCPUFAs and EFAs by BeWo cells
compared with preimmune serum. Together, these results show
the preferential uptake of LCPUFAs by the placenta that is most
probably mediated via the p-FABPpm.
Am J Clin Nutr
2000;71(suppl):315S–22S.
316S
DUTTA-ROY
the interaction of EFAs and LCPUFAs with isolated human placental membranes and with human placental choriocarcinoma
(BeWo) cells. The aim of this article is to bring together recent
developments on the placental uptake and metabolism of LCPUFAs
in humans and to advance concepts concerning the role of the
newly discovered placental plasma membrane fatty acid binding
proteins (p-FABPpm) in these processes (15, 16).
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MATERNAL METABOLISM AND TRANSPORT OF EFAS
AND THEIR LCPUFA DERIVATIVES
Linoleic acid (LA; 18:2n26) and a-linolenic acid (ALA;
18:3n23) are the 2 main dietary EFAs. LA and ALA are not
interchangeable, but can be further elongated and desaturated by
the same enzyme systems to produce n26 and n23 LCPUFAs in
the body (1, 17). Whereas dietary LA and ALA are primarily
from vegetable oils, preformed LCPUFAs may also be consumed
in foods of animal origin. The importance of LCPUFAs has been
related to their structural action, their specific interaction with
membrane proteins, and their ability to serve as precursors of
second messengers (1, 18, 19). Therefore, LA and ALA must be
converted to their metabolites, LCPUFAs, to exert their full
range of biological actions (1). Maternal EFA metabolism is crucial for fetal growth and development because the fetus depends
on maternal EFAs and LCPUFAs (4, 15). However, it is important to note that the accretion of LCPUFAs [eg, AA, eicosapentaenoic acid (EPA; 20:5n23), DHA, and dihomo-g-linolenic
acid (20:3n26)] cannot be quantitatively explained by desaturation and elongation of dietary EFAs alone (1). Relations between
dietary EFAs and fatty acid profiles of tissues are complex and
appear to be dependent on many factors and underlying
processes (1). Except for DHA, LCPUFAs are substrates for
cyclooxygenase (prostaglandin-endoperoxide synthase) and
lipoxygenases and produce a variety of compounds collectively
called eicosanoids (1). These compounds have diverse biological
functions in cell growth and development, inflammation, and the
cardiovascular system (1, 3, 18, 19). DHA is involved mainly in
retina and brain function, but not through the eicosanoid pathways (4, 20). The biological response elicited after eicosanoid
release is dependent on the net balance of eicosanoids derived
from n26 and n23 LCPUFAs. In the Western diet, AA is the
most predominant precursor fatty acid of the highly biologically
active eicosanoids of the 2 series (ie, eicosanoids with double
bonds). AA thus provides adequate substrates for synthesis of
circulating vasoactive factors such as prostacyclin and thromboxane A2 (1, 21). The ratio of thromboxane A2 to prostacyclin
is an index of the relative activity of the opposing stimuli that
modulate vascular tone and platelet activation (1, 21). The ratio
of thromboxane A2 and prostacyclin in intervillous space may be
involved in the mechanism of initiating myometrial contraction
during labor. In addition, the endogenous formation of cyclooxygenase and noncyclooxygenase metabolites of AA has been
implicated in the expression of the early response genes c-fos,
Egr-1, c-myc, and c-jun in different cell types after mitogenic
stimulation; EPA, however, inhibits the expression of these genes
(3). Therefore, supplemental EPA not only decreases AA availability but also competes with AA for cyclooxygenase and
lipoxygenase enzymes and thereby reduces the formation of the
growth-stimulatory eicosanoids (1, 3).
Because both n23 and n26 fatty acids are present in vertebrate
tissues owing only to dietary intake, important physiologic conse-
quences follow the inadvertent selection of different average daily
dietary supplies of these 2 types of EFAs. Because each type of
EFA can interfere with the metabolism of the other, an excess of
n26 fatty acids will reduce the metabolism of ALA, possibly leading to a deficit of n23 LCPUFA metabolites. This is a matter of
concern in infant formulas, which may contain an excess of LA
without balancing n23 fatty acids. Therefore, a proper balance
between the n26 and n23 fatty acids in the diet is important to
maintain optimum growth and development, and there is an
emerging consensus that infant formulas should be supplemented
with DHA rather than EPA and have adequate amounts of AA.
However, it is not known how the pregnant mother is able to provide an adequate supply of LCPUFAs, especially during the last
trimester of pregnancy when the fetus requires large amounts of
both AA (n26) and DHA (n23), but not much EPA (n23).
Several studies (10, 11, 15, 22) of the fatty acid composition
of fetal and maternal plasma showed that at birth, LA represents
<10% of the total fatty acids in cord plasma compared with 30%
in maternal plasma but, surprisingly, the AA concentration of
cord plasma is twice (<10%) that observed in the mother (5%).
Similarly, the ALA concentration in newborns is half of that in
the mother (0.3% and 0.6%, respectively), whereas the DHA
concentration is double (3% and 1.5%, respectively). EPA concentrations were low, with no significant difference between
fetuses and mothers. This situation, in which the relative plasma
concentrations of the n23 and n26 LCPUFAs exceeds those of
their precursors, is specific to newborns and has never been
observed in adults. It is obviously an extremely favorable situation for the development of the newborn, especially at a time
when large quantities of AA and, particularly, DHA are needed
by the brain and retina. However, 3 important questions arise:
1) What are the underlying mechanisms leading to this situation
at birth? 2) How does the situation evolve throughout gestation?
and 3) How are the n23 and n26 LCPUFAs (DHA and AA)
preferentially transferred?
Although the delivery of fatty acids from the maternal circulation to the developing fetus has been studied extensively (4,
23) and various suggestions have been put forward, none of the
above questions have been answered satisfactorily. Because
human placental tissue lacks both the D6- and D5-desaturase
activities (4, 23–25), any LCPUFAs in the fetal circulation must
be derived primarily from the maternal plasma. However, the situation has become more complicated by the discovery of desaturase and elongase activity in developing fetal brain and liver
(24), although it is not certain to what extent desaturation and
elongation of transferred linoleic and a-linolenic acids may contribute to fetal LCPUFAs.
In the fetomaternal unit, free fatty acids (FFAs) originating in
the maternal circulation are the major source of fatty acids for
transport across the placenta (4, 23–25) because triacylglycerols
are not transported intact (4, 26, 27). Maternal lipoprotein lipase
must be active to facilitate placental uptake of FFAs from circulating triacylglycerols but not from chylomicrons (4, 15).
Recently, we showed that purified placental microvillous membranes exhibit 2 distinct triacylglycerol hydrolase activities: a
minor activity at pH 8.0 and a major activity at pH 6.0 (28). The
triacylglycerol hydrolase activity at pH 8.0 appears to be
lipoprotein lipase (identified by important criteria such as serum
stimulation and salt inhibition), whereas the activity at pH 6.0 is
unique because it is almost abolished by serum but is not
affected by high sodium chloride concentrations. Therefore, it is
FATTY ACID TRANSPORT ACROSS THE PLACENTA
TABLE 1
Fatty acid binding specificity of human placental membranes1
[14C]Fatty acid plus
unlabeled ligand (20-fold)
Inhibition of
[14C]fatty acid binding
%
40
35
90
95
90
5
3
41
60
70
68
50
6
4
45
58
75
28
30
3
2
1
Binding specificity of each [14C]fatty acid to placental membranes was
determined by conducting incubations in the presence of a 20-fold excess of
unlabeled ligands (adapted from reference 30). The percentage inhibition of
[14C]fatty acid binding to membranes was calculated assuming the total
binding of [14C]fatty acid to membranes in the absence of unlabeled ligands.
possible that the inhibitory effect of serum on triacylglycerol
hydrolase activity at pH 6.0 in placental microvillous membranes may not allow this enzyme to act on maternal lipoproteins
but only on intracellular triacylglycerol stores in the placenta. If
true, then this membrane-bound triacylglycerol hydrolase (optimum pH 6.0) may play an important role in releasing FFAs by
hydrolyzing the placental triacylglycerol stores. This unique triacylglycerol hydrolase activity at pH 6.0 therefore may be
involved in the packaging, release, or both of FFAs from the placenta. However, lipoprotein lipase, which is present mostly in
placental macrophages, may be responsible for hydrolysis of
maternal plasma lipoproteins, as suggested by Bonet et al (29).
Lipoprotein receptors, mainly responsible for the uptake of cholesterol required for fetal tissue synthesis, have been detected on
both placental macrophages and syncytiotrophoblasts (15). They
may also be involved in fatty acid uptake by the placenta. However, the metabolic fate of fatty acids transported via lipoprotein
receptors is uncertain and may not account for the preferential
accumulation of LCPUFAs in fetal tissues.
PREFERENTIAL UPTAKE OF LCPUFAS BY PLACENTAL
MEMBRANES
The preferential accumulation of LCPUFAs in fetal tissues was
initially thought to be the result of the combined effects of placental fatty acid uptake from the maternal plasma, selective fatty
acid oxidation, lipid synthesis in the placenta, and LCPUFA synthesis by the fetal liver (4, 15). Evidence on the role of the placenta in preferential accumulation of LCPUFAs in fetal tissues has
emerged from research in our laboratory in which the fatty acid
binding characteristics of human placental membranes were determined by using 4 different radiolabeled fatty acids [LA, ALA, AA,
and the nonessential fatty acid oleic acid (18:1n29)] (30).
The binding of both EFAs and LCPUFAs to human placental
membranes was highly reversible and specific compared with that
of oleic acid (30). In addition, oleic acid binding was inhibited
strongly by the LCPUFAs [AA, g-linolenic acid (18:3n26), and
EPA], followed by the relevant parent EFAs (LA and ALA). The
lack of strong inhibition of the binding of EFAs and LCPUFAs to
placental membranes by oleic acid suggested the existence of
stronger affinities for EFAs and LCPUFAs compared with oleic
acid (30). EPA and its eicosanoid metabolites are considered to be
growth inhibitors because they reduce the availability of AA and
its metabolites by competing for cyclooxygenase, lipoxygenase,
and elongation and desaturation pathways of EFA metabolism
(1, 3). EPA has little inhibitory effect on AA transport by placental membranes. In contrast with EPA, its parent fatty acid, ALA,
strongly inhibited the binding of both AA and LA. (Table 1).
Non–fatty acid ligands such as bromsulfophthalein and a-tocopherol did not inhibit the binding of any of the radiolabeled fatty
acids, indicating that binding sites are specific to fatty acids. The
competition experiments also suggested that the binding sites have
heterogeneous binding affinities for different fatty acids. Binding
sites seem to have a strong preference for LCPUFAs: the order of
competition was AA >> LA > ALA >> oleic acid. Our finding on
the preferential binding of LCPUFAs by placental membranes was
supported by Haggarty et al (31), who also showed that the human
placenta can selectively transfer LCPUFAs and EFAs to the fetal
circulation in preference to the nonessential fatty acids.
Our data also suggested that trans fatty acids compete with
LCPUFAs for fatty acid binding sites in human placental membranes, thereby inhibiting the transport of EFA and LCPUFAs to
the placenta (30). trans Fatty acids are not synthesized in situ but
are present in both fetal and placental tissues, which suggests
that they are transported through the placenta from the maternal
circulation (32, 33). Our studies support these observations that
maternal trans fatty acids can be transported to the fetal circulation via the placenta. Once trans fatty acids are in the fetoplacental unit they may exert detrimental effects on growth and
development by disturbing LCPUFA metabolism (eicosanoid
production) and membrane structure and function (32, 33).
IDENTIFICATION AND CHARACTERIZATION OF A
PLACENTAL MEMBRANE FATTY ACID BINDING
PROTEIN
The tissue uptake of FFAs has been studied extensively and a
variety of mechanisms of FFA uptake have been proposed,
including passive diffusion, specific binding to plasma membrane associated fatty acid binding proteins such as ubiquitous
FABPpm, fatty acid translocase, and fatty acid transport protein
(FATP) (34–38). There are now several reports providing evidence of the involvement of these membrane proteins in the
uptake of FFAs into various mammalian tissues (34). Therefore,
the presence of a membrane FABP in the placenta was sought.
We identified and purified p-FABPpm from human and sheep placentas by using a technique similar to that used for ubiquitous
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[14C]Linoleic acid (18:2n26) plus unlabeled
Oleic acid (18:1n29)
a-Linolenic acid (18:3n23)
g-Linolenic acid (18:3n26)
Arachidonic acid (20:4n26)
Eicosapentaenoic acid (20:5n23)
a-Tocopherol
Bromsulfophthalein
[14C]a–Linolenic acid (18:3n23) plus unlabeled
Oleic acid (18:1n29)
Linoleic acid (18:2n26)
g-Linolenic acid (18:3n26)
Arachidonic acid (20:4n26)
Eicosapentaenoic acid (20:5n23)
a-Tocopherol
Bromsulfophthalein
[14C]Arachidonic acid (20:4n26) plus unlabeled
Oleic acid (18:1n29)
Linoleic acid (18:2n26)
g-Linolenic acid (18:3n26)
a-Linolenic acid (18:3n23)
Eicosapentaenoic acid (20:5n23)
a-Tocopherol
Bromsulfophthalein
317S
318S
DUTTA-ROY
ROLE OF p-FABPpm IN PREFERENTIAL UPTAKE OF
LCPUFAS BY THE PLACENTA
To elucidate further the role of p-FABPpm in the preferential
transfer of maternal plasma LCPUFAs across the human placenta, we investigated the direct binding of the purified p-FABPpm
with various fatty acids (41). Binding of radiolabeled fatty acids
to the purified p-FABPpm revealed that the placental protein had
higher affinities (Kd) and binding capacities (Bmax) for LCPUFAs
than for other fatty acids. The apparent Bmax values for oleic
acid, LA, AA and DHA were 2.0, 2.1, 3.5, and 4.0 mol/mol
FABPpm, whereas the apparent Kd values were 1.0, 0.73, 0.45,
and 0.40 mmol/L, respectively (41). There was no significant difference in Kd and Bmax values between oleic acid and LA or
between AA and DHA. However, there were significant differ-
ences in these values between the LCPUFAs (AA and DHA) and
oleic acid and LA. Human serum albumin did not show such
variations in binding capacities and affinities for these fatty
acids; the Kd and Bmax values for these fatty acids were <1 mmol/L
and 5 mol/mol FABPpm, respectively (41).
During the last trimester of pregnancy, plasma FFA concentrations increase rapidly to support the fetal demand for LCPUFAs
(4, 23–25). Therefore, we also examined the binding of a fatty
acid mixture taken from women during the last trimester of
pregnancy that mimicked that of plasma with the purified
p-FABPpm. The total FFA concentration in the plasma pool
(x– ± SD, in mmol/L) was 0.71 ± 0.015, of which 0.22 ± 0.07 was
palmitic acid, 0.14 ± 0.08 was stearic acid, 0.19 ± 0.06 was
oleic acid, 0.12 ± 0.021 was LA, 0.011 ± 0.004 was ALA,
0.016 ± 0.005 was AA, and 0.015 ± 0.003 was DHA (42). Thus,
76.7% of total fatty acids were nonessential (palmitic, stearic,
and oleic acids) and 23% were essential and, of these, LCPUFAs
amounted to 4.5%. AA and DHA were present in similar concentrations in the plasma FFA pool of pregnant women despite a
10-fold difference in concentrations of their n26 and n23 precursors, LA and ALA, respectively. This suggests a mechanism
that increases the availability of unesterified DHA in the plasma
of pregnant women and thus increase the fetal supply of DHA.
p-FABPpm bound only 14.58% of the total fatty acids when the
purified protein was incubated with a fatty acid mixture as
described above (Table 2). Despite the high concentrations of the
nonessential fatty acids in the assay mixture, little stearic or oleic
acid bound to the p-FABPpm (0.57 ± 0.07 and 4.05 ± 0.40 nmol,
respectively; n = 3) whereas binding of palmitic acid was not
detectable. For the 2 EFAs, binding of ALA was not detected,
whereas almost 23% of the LA present in the mixture bound to
the protein. In contrast, 98% of the AA and 83% of the DHA
present in the assay mixture was bound to the protein
(1.62 ± 0.04 and 1.24 ± 0.03 nmol, respectively; n = 3). Human
serum albumin, with a similar ratio of fatty acid to protein,
bound 2.5-fold more fatty acids than did the p-FABPpm with no
preference for any particular type of fatty acid. The percentage
of each fatty acid bound to the human serum albumin was greatest for stearic acid (71%) followed by oleic acid (34%), palmitic
acid (33%), and least for LA (18%) and AA (23%); binding of
DHA was not detectable. These data clearly show that p-FABPpm
preferably binds with LCPUFAs, whereas human serum albumin
does not show any preference for particular fatty acids present in
the plasma FFA pool of pregnant women during the last trimester
TABLE 2
Binding of mixture of fatty acids as present in the plasma free fatty acid pool of pregnant women to placental plasma membrane fatty acid binding protein
(p-FABPpm) and in human serum albumin1
Fatty acid bound
Incubation mixture
p-FABPpm
nmol
Human serum albumin
mol/mol protein
Palmitic acid (16:0)
51.00
ND
Stearic acid (18:0)
35.00
0.57 ± 0.07
Oleic acid (18:1n29)
49.00
4.05 ± 0.40
Linoleic acid (18:2n26)
30.00
2.76 ± 0.23
a-Linolenic acid (18:3n23)
2.94
ND
Arachidonic acid (20:4n26)
4.20
1.62 ± 0.04
Docosahexaenoic acid (22:6n23)
3.70
1.24 ± 0.03
1–
x ± SD of 3 separate experiments in which triplicate determinations were performed. ND, not detectable. From reference 42.
2,3
Significantly different from binding of p-FABPpm (two-tailed Student’s t test), 2 P < 0.01, 3 P < 0.05.
6.89 ± 0.43
9.96 ± 0.332
6.58 ± 0.263
2.21 ± 0.17
ND
0.39 ± 0.08
ND
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FABPpm (16, 39). The apparent molecular mass of the p-FABPpm
was estimated to be <40 kDa, as determined by gel permeation
chromatography and by sodium dodecyl sulfate–polyacrylamide
gel electrophoresis (16). The isoelectric point (pI) value and
amino acid composition of p-FABPpm (40 kDa) are different
from those of ubiquitous FABPpm (40 kDa), fatty acid translocase (88 kDa), and FATP (62 kDa) (34–39). In addition, unlike
ubiquitous FABPpm (40), p-FABPpm does not have glutamic
oxaloacetic transaminase activity. Therefore, despite their similar size and membrane location (both are peripheral-membranebound proteins), p-FABPpm and ubiquitous FABPpm differ in both
structure and function. The autoradioblotting technique, which
was initially developed to detect and estimate cellular retinolbinding proteins, was used to investigate the [14C]fatty acid binding activity of the purified membrane protein and also to detect
p-FABPpm in solubilized placental membranes (16). Evidence for
the involvement of p-FABPpm in fatty acid uptake came from the
trypsin-treated placental membranes, in which specific [14C]fatty
acid binding decreased compared with untreated membranes
(30). Furthermore, preincubation of human placental membranes
with polyclonal antiserum against the human p-FABPpm inhibited the binding of fatty acids but the degree of inhibition varied
depending on the type of fatty acid (30). Antibody-mediated
inhibition was much more effective for the LCPUFA and EFA
binding than the oleic acid binding. This suggests again that fatty
acid binding is heterogeneous, and that p-FABPpm may be preferentially involved in the uptake of EFAs and their LCPUFA
derivatives.
FATTY ACID TRANSPORT ACROSS THE PLACENTA
TABLE 3
Effect of fatty acid addition on uptake of oleic and linoleic acids by
human placental choriocarcinoma (BeWo) and HepG2 liver cells1
Radiolabeled fatty acid
plus unlabeled fatty acid
Fatty acid uptake
BeWo cells
HepG2 cells
mmol/g protein
[ H]Oleic acid (18:1n29)
5.36 ± 0.14
22.89 ± 0.34
[3H]Oleic acid plus linoleic
acid (18:2n26) (10-fold)
1.41 ± 0.152
8.67 ± 0.483
[14C]Linoleic acid
6.72 ± 0.50
27.78 ± 1.59
[14C]Linoleic acid plus
oleic acid (10-fold)
5.40 ± 0.30
16.83 ± 1.892
[14C]Linoleic acid plus a-linolenic
acid (18:3n23) (10-fold)
1.14 ± 0.094
17.49 ± 0.362
1–
x ± SEM of 3 separate experiments in which triplicate determinations
3
of pregnancy. It has been generally accepted that serum albumin
serves as a reservoir of fatty acids for replenishment of those
taken up by cells. The binding experiments provide direct evidence for the role of p-FABPpm in preferential sequestration of
maternal LCPUFAs by the placenta for transport to the fetus.
With the exclusive location of the p-FABPpm on the on the maternal-facing membranes of the placenta (43) and its preference for
LCPUFAs in the plasma FFA pool, a unidirectional flow of
maternal LCPUFAs to the fetus is favored.
UPTAKE AND METABOLISM OF FATTY ACIDS IN A
HUMAN PLACENTAL CHORIOCARCINOMA CELL LINE
All the above-mentioned studies were performed using isolated human placental membranes and therefore no information
was available on intact trophoblast cells with metabolic activity.
We therefore examined the uptake and metabolism of oleic acid,
linoleic acid, AA, and DHA in the BeWo human placental choriocarcinoma cell line (44). We also compared the uptake and metabolism of oleic acid and LA by BeWo cells with the carrier-mediated uptake by HepG2 liver cells (45). These 2 cell types are
derived from organs with specialized roles in lipid metabolism.
For example, the liver is involved in a multiplicity of lipid metabolic roles including synthesis, esterification, lipoprotein export,
and oxidation for energy, whereas, as already noted, the placenta
is primarily concerned with sequestering the maternal LCPUFAs
and transporting them to the fetus.
The kinetic data and temperature-dependent nature of fatty acid
uptake by BeWo cells indicated a saturable process. Oleic acid
was taken up least and DHA most by these cells. Moreover, competitive studies indicated a preferential uptake compared with
oleic acid in the order of DHA, AA, and LA by BeWo cells (44),
supporting previous studies on the placental transport of DHA
(46). In contrast, HepG2 cells did not exhibit a differential affinity for the fatty acids studied (Table 3).
With the use of a polyclonal antibody to p-FABPpm, Western blot
analyses showed the presence of a p-FABPpm in BeWo cells, but not
in HepG2 cells. Ubiquitous FABPpm in HepG2 cells did not react
with p-FABPpm, further showing the difference between ubiquitous and p-FABPpm (44). Cells pretreated with p-FABPpm antiserum
inhibited uptake of oleic acid, LA, AA, and DHA to different
degrees than did untreated cells (45). With the same amount of antiserum, oleic acid uptake was inhibited least (32%), whereas uptake
of AA (68%) and DHA (64%) was inhibited most, followed by LA
(50%) (45). These results clearly show that p-FABPpm may be
involved in the preferential uptake of EFAs and their LCPUFA
derivatives by these cells.
We also compared the metabolism of oleic acid, LA, AA, and
DHA in BeWo cells and observed marked differences (47).
BeWo cells were found to rapidly incorporate exogenous AA
and DHA into the total cellular lipid pool. The extent of esterification was more rapid for DHA than for AA, although this difference abated with time, leaving only a small percentage of the
fatty acids in their unesterified form. In the cellular lipids, these
fatty acids were esterified mainly into the phospholipid and the
triacyglycerol fractions; smaller amounts were also detected in
the diacylglycerol and cholesteryl ester fractions. Almost 60%
of the total DHA taken up by cells was esterified into triacylglycerol whereas 37% was in phospholipid fractions. The
reverse was true for AA; 60% of the total uptake was incorporated into phospholipid fractions whereas < 35% was in triacyglycerol fractions. Marked differences were also found in the distribution of the fatty acids into individual phospholipid classes.
The higher incorporations of DHA and AA were found in phosphatidylcholine and phosphatidylethanolamine, respectively.
The greater cellular uptake of DHA and its preferential incorporation into triacyglycerols suggests that the uptake and transport modes of DHA by the placenta to the fetus are different
from those of AA. The preferential incorporation of DHA into
triacylglycerol suggests that triacylglycerol may play an important role in the placental transport of DHA to the fetal circulation. Together, these results show the preferential uptake of
LCPUFAs by BeWo cells, which is most probably mediated via
p-FABPpm. The uptake and subsequent esterification of DHA
was the greatest and the esterification of DHA into triacylglycerol was more efficient than that of the other 3 fatty acids. One
of the main functions of the placenta is to deliver DHA into the
fetal circulation; therefore, the triacylglycerol form may favor
the transport of DHA to the fetal circulation. However, at present it is not known how and in what form DHA is released from
the placenta into the fetal circulation.
A multistep process of cellular uptake and intracellular translocation of FFAs facilitated by various membrane-associated and
cytoplasmic FABPs has been proposed (36). We therefore examined the presence of FABPs in primary trophoblasts, BeWo cells,
and placental membranes (both microvillous and basal). Western
blot analysis showed the presence of multiple membrane-associated and cytoplasmic FABPs in human placental cells (48). In
addition to p-FABPpm, fatty acid translocase (88 kDa) and FATP
(62 kDa) were detected in both microvillous and basal membranes of the human placenta (48). Among the cytoplasmic
FABPs, heart- (H) and liver- (L) type FABPs were detected in the
cytosol of human placental primary trophoblasts as well as in
BeWo cells (48). Immunoreactivity of epidermal- (E) type FABP
was not detected in trophoblasts or BeWo cells despite its presence in human placental cytosol. The location of fatty acid
translocase and FATP on both sides of bipolar placental cells may
favor transport of FFA pools in both directions, ie, from the
mother to the fetus and vice versa. However, p-FABPpm, because
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were performed. Competition between [3H]oleic acid and [14C]linoleic acid
for uptake by BeWo and HepG2 cells was carried out in the presence of and
in the absence of a 10-fold excess of unlabeled fatty acid. From reference
45, and FM Campbell and AK Dutta-Roy, unpublished observations, 1997.
2–4
Significantly different from control cells (with no inhibitors) (twotailed Student’s t test): 2 P < 0.0001, 3 P < 0.005, 4 P < 0.001.
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of its exclusive location on the microvillous membranes, may
favor the unidirectional flow of maternal plasma LCPUFAs present in the plasma FFA pool to the fetus because of the binding
specificity for LCPUFAs. Although the roles of these proteins in
placental fatty acid uptake and metabolism are yet to be fully
understood, their complex interaction may be involved in the
uptake of maternal FFAs by the placenta for delivery to the fetus.
CONCLUSIONS
Among the various biofactors released at the fetomaternal
interface, LCPUFAs play major roles in fetoplacental development and pregnancy outcome (36, 49). LCPUFAs are important for optimal growth because they are the precursors of
eicosanoids and also maintain cell membrane structure and function (1). The preferential accumulation of maternal LCPUFAs in
fetal tissues has been thought to be the result of combined
processes occurring in both the mother and the fetoplacental
unit. Our data indicate that p-FABPpm, located exclusively on the
maternal-facing membranes of the placenta, may be involved in
the sequestration of maternal LCPUFAs and therefore may play
a critical role in fetal growth and development (36, 49). However, further studies are required on the fatty acid–binding
domain of the p-FABPpm, which allows the preferential binding
of LCPUFAs over nonessential fatty acids. The preferential
incorporation of DHA into the triacylglcyerol fraction suggests
that triacylglcyerols may play an important role in the placental
transport of DHA to the fetal circulation. The major membrane
lipase activity at pH 6.0 may play an important role in packaging
placental-stored DHA for fetal transport.
Although the exact roles of the membrane-associated FABPs
(fatty acid translocase, FATP, and FABPpm) in complex FFA
uptake and metabolism in the human placenta are not yet understood, studies in other tissues suggest that these proteins alone or
in tandem may be involved in effective uptake of FFAs (50).
Location of fatty acid translocase and FATP on both sides of the
bipolar placental cells may allow bidirectional flow of all fatty
acids (nonessential, essential, and long-chain polyunsaturated)
across the placenta, whereas the exclusive location of p-FABPpm
on the maternal side may favor the unidirectional flow of maternal LCPUFAs to the fetus by virtue of preference for these fatty
acids (48).
Cytoplasmic heart- and liver-type FABPs in the placenta have
been observed (48). There are significant differences in the properties and regulation of these 2 FABP types. Heart FABP binds
only long-chain fatty acids whereas liver FABP binds heterogeneous ligands such as bile salts, heme, peroxisome proliferators,
selenium, lysophosphatidic acid, and eicosanoids (36). By virtue
of its binding of eicosanoids, liver FABP may play a critical role
in eicosanoid synthesis in the fetoplacental unit. Liver FABP
increases fatty acid uptake and targets fatty acids for esterification into phospholipids. It has been suggested that FABPs may
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FIGURE 1. Putative roles of the placental-membrane-associated and cytoplasmic fatty acid binding proteins (FABPs) in placental fatty acid uptake
and metabolism. The location of fatty acid translocase and FAPBs on both sides of the bipolar placental cells and the lack of specificity for particular
types of free fatty acids (FFAs) allow transport by FAPBs of all FFAs (nonessential, essential, and long-chain polyunsaturated) bidirectionally, ie, from
the mother to the fetus and vice versa. However, by virtue of its exclusive location on the maternal-facing placental membranes and preference for longchain polyunsaturated fatty acids (LCPUFAs), placental plasma membrane FAPB (p-FABPpm) sequesters maternal plasma LCPUFAs to the placenta.
Cytoplasmic FABPs may be responsible for transcytoplasmic movement of FFAs to their sites of esterification, b-oxidation, or to the fetal circulation
via placental basal membranes. FAT, fatty acid translocase; FATP, fatty acid transport protein; L-, liver-type; H-, heart-type; PG, prostaglandins; LT,
leukotrienes; TG, triacylglycerols, PL, phospholipids; CE, cholesteryl esters; HEPTE, hydroperoxyeicosatraenoic acid. Reprinted from Placenta, Vol
19, (5/6), Campbell FM, Bush PG, Veerkamp JH, Dutta-Roy AK, Detection and cellular localization of plasma membrane–associated and cytoplasmic
fatty acid binding proteins in human placenta, pp409–15, 1998, by permission of the W B Saunders Company Limited (48).
FATTY ACID TRANSPORT ACROSS THE PLACENTA
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