Asim K. Duttaroy

Transport mechanisms for long-chain polyunsaturated fatty acids in the human placenta

To understand the placental role in the processes responsible for the preferential accumulation of maternal long-chain 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 (p-FABP(pm)) with a molecular mass of approximately 40 kDa was identified. The purified p-FABP(pm) 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-FABP(pm) 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-FABP(pm).

Transport of fatty acids across the human placenta: a review.

Essential fatty acids and their long chain polyunsaturated fatty acid derivatives (20C) such as docosahexaenoic and arachidonic acids are critical for proper fetal growth and development. Dietary intake as well as metabolism of these fatty acids, and their subsequent transfer from the mother to the fetus are therefore important requisites for developing fetus. The placenta is the key organ through which nutrients such as these fatty acids flow from the mother to the fetus. Cellular uptake and translocation of long chain fatty acids (LCFAs) in different tissues is achieved by a concert of co-existing mechanism. Although LCFA can enter the cell via passive diffusion, emerging reports indicate that LCFA uptake is tightly regulated by several plasma membrane-located transport/binding proteins such as fatty acid translocase (FAT/CD36), plasma membrane fatty acid binding protein (FABPpm), fatty acid transport protein (FATP) and intracellular FABPs in several tissues including human placenta. Fatty acid activated transcription factors (PPARs, LXR, RXR, and SREBP-1) have been demonstrated to regulate these fatty acid transport/binding proteins, and placental functions. Maternal fatty acids therefore may regulate their own placental transport as well as placental function via several fatty acid-activated transcription factors. This review summarizes recent developments on placental fatty acid transport and metabolisms, and the regulatory roles of these proteins in these processes.

Detection and cellular localization of plasma membrane-associated and cytoplasmic fatty acid-binding proteins in human placenta

The aim of this study was to investigate location and the types of membrane-associated and cytoplasmic fatty acid-binding proteins in human placental trophoblasts using monospecific polyclonal antibodies. Western blot analysis demonstrated the presence of multiple membrane and cytoplasmic fatty acid transport/binding proteins in human placenta. In addition to previously reported placental membrane fatty acid-binding (p-FABPpm, 40 kDa), fatty acid translocase (FAT, 88 kDa) and fatty acid transport protein (FATP, 62 kDa) were detected in both microvillous and basal membranes of the human placenta. Among the cytoplasmic proteins, heart (H) and liver (L) type FABP were detected in the cytosol of the human placental primary trophoblasts as well as in human placental choriocarcinoma (BeWo) cells. The immunoreactivity of epidermal type (E)-FABP was not detected in trophoblasts or BeWo cells despite its presence in human placental cytosol. Location of FAT and FATP on the both sides of the bipolar placental cells may favour transport of free fatty acids (FFA) pool in both directions i.e. from the mother to the fetus and vice versa. However, p-FABPpm, because of its exclusive location on the microvillous membranes, may favour the unidirectional flow of maternal plasma long-chain polyunsaturated fatty acids present in the FFA pool to the fetus, due to binding specificity for these fatty acids. Although the roles of these proteins in placental fatty acid uptake and metabolism are yet to be understood fully, their complex interaction may be involved in the uptake of maternal FFA by the placenta for delivery to the fetus.

Preferential uptake of long chain polyunsaturated fatty acids by isolated human placental membranes

Fatty acid uptake by the placenta is thought to be a carrier-mediated process, however the mechanism by which long chain polyunsaturated fatty acids (LCPUFA) are preferentially accumulated from the maternal circulation to the fetal tissues is still unclear. To examine the role of the placenta in this process, binding of four different radiolabelled fatty acids ([14C]oleate, [14C]linoleate, [14C]a-linolenate and [14C]arachidonate) to human placental membranes was studied. Binding of fatty acid was found to be time- and temperature dependent. At equilibrium, the total binding of oleate was highest (5.1 ± 0.1 nmoles/mg protein) followed by linoleate (2.8 ± 0.31 nmoles/mg protein) and arachidonate (2.06 ± 0.4 nmoles/mg protein) and α-linolenate binding was lowest (0.5+0.1 nmoles/mg protein). However, oleate had the lowest specific binding (37% of the total binding) whereas arachidonate had the highest specific binding (∼ 86% of the total binding) followed by linoleate and a-linolenate (62%, and 69% of the total binding, respectively). Binding of each [14C] fatty acid was also assessed in the presence of 20-fold excess of other unlabelled ligands. Binding sites seem to have preference for the binding of [14C] fatty acids in the following order: arachidonic acid ⋙ linoleic acid ≫ a-linolenic acid ⋙≫ oleic acid, whereas BSP and a-tocopherol did not show any competition with any of the [14C] fatty acids. These data suggest that the fatty acid binding sites in placental membranes are specific for the fatty acids but that they have heterogeneous affinities.Trans fatty acids (elaidic and linoelaidic acids) also competed very strongly for the [14C] fatty acid binding. Polyclonal antiserum raised against placental FABPpm inhibited binding of these [14C]fatty acids but with variable degrees of inhibition; EFA/LCPUFA binding was much more than that of oleate. Our data suggest that EFA/LCPUFA bound to albumin are preferentially transported by human placental membranes and that the placental FABPpm may be involved in the sequestration of EFA/LCPUFA by the placenta.

Effects of kiwi fruit consumption on platelet aggregation and plasma lipids in healthy human volunteers

Fruits and vegetables have been thought to be beneficial in cardiovascular disease. The beneficial effects of fruits and vegetables may be explained by the antioxidants and other components contained therein. These nutrients may function individually or in concert to protect lipoproteins and vascular cells from oxidation, or by other mechanisms such as reducing plasma lipid levels (LDL cholesterol, triglycerides), and platelet aggregation response. Kiwi fruit which contains high amounts of vitamin C, vitamin E and polyphenols may be beneficial in cardiovascular disease; however very little is known about its cardioprotective effects. Platelets are involved in atherosclerotic disease development and the reduction of platelet activity by medications reduces the incidence and severity of disease. To this end, we evaluated whether consuming kiwi fruit modulated platelet activity and plasma lipids in human volunteers in a randomized cross-over study. We report that consuming two or three kiwi fruit per day for 28 days reduced platelet aggregation response to collagen and ADP by 18% compared with the controls (P<0.05). In addition, consumption of kiwi fruit lowered blood triglycerides levels by 15% compared with control (P<0.05), whereas no such effects were observed in the case of cholesterol levels. All these data indicate that consuming kiwi fruit may be beneficial in cardiovascular disease.

Placental membrane fatty acid-binding protein preferentially binds arachidonic and docosahexaenoic acids.

To elucidate further the role of placental membrane fatty acid-binding protein (p-FABPpm) in preferential transfer of maternal plasma long chain polyunsaturated fatty acids (LCPUFA) across the human placenta, direct binding of the purified protein with various radiolabelled fatty acids (docosahexaenoic, arachidonic, linoleic and oleic acids) was investigated. Binding of these fatty acids to the protein revealed that p-FABPpm had higher affinities and binding capacities for arachidonic and docosahexaenoic acids compared with linoleic and oleic acids. The apparent binding capacities (Bmax) values for oleic, linoleic, arachidonic and docosahexaenoic acids were 2.0 +/- 0.14, 2.1 +/- 0.17, 3.5 +/- 0.11, 4.0 +/- 0.10 mol per mol of p-FABPpm whereas the apparent dissociation constant (Kd) values were 1.0 +/- .0.07, 0.73 +/- 0.04, 0.45 +/- 0.03 and 0.4 +/- 0.02 microM, respectively (n=3). In the case of human serum albumin, the Kd and Bmax values for all fatty acids were around 1 microM and 5 mol/mol of protein, respectively. These data provide direct evidence for the role of p-FABPpm in preferential sequestration of maternal arachidonic and docosahexaenoic acids by the placenta for transport to the fetus by virtue of its preferential binding of these fatty acids.

Effects of tomato extract on human platelet aggregation in vitro

Among all fruits tested in vitro for their anti-platelet property, tomato had the highest activity followed by grapefruit, melon, and strawberry, whereas pear and apple had little or no activity. Tomato extract (20-50 w l of 100% juice) inhibited both ADP- and collagen-induced aggregation by up to 70% but could not inhibit arachidonic acid-induced platelet aggregation and concomitant thromboxane synthesis under similar experimental conditions. The anti-platelet components (MW <1000 Da) in tomatoes are water soluble, heat stable and are concentrated in the yellow fluid around the seeds. The active fractions were separated using gel filtration and HPLC. The aqueous fraction (110 000 2 g supernatant) of tomatoes containing anti-platelet activity was subjected to gel filtration column chromatography (Biogel P2 column). The activity was fractionated into two peaks, peak-3 and peak-4 (major peak). Subsequently, peak-4 was further purified by HPLC using a reversed-phase column. NMR and mass spectroscopy studies indicated that peak F2 (obtained from peak 4) contained adenosine and cytidine. Deamination of peak F2 with adenosine deaminase almost completely abolished its anti-platelet activity, confirming the presence of adenosine in this fraction. In comparison, deamination of peak-4 resulted in only partial loss of inhibitory activity while the activity of peak-3 remained unaffected. These results indicate that tomatoes contain anti-platelet compounds in addition to adenosine. Unlike aspirin, the tomato-derived compounds inhibit thrombin-induced platelet aggregation. All these data indicate that tomato contains very potent anti-platelet components, and consuming tomatoes might be beneficial both as a preventive and therapeutic regime for cardiovascular disease.Among all fruits tested in vitro for their anti-platelet property, tomato had the highest activity followed by grapefruit, melon, and strawberry, whereas pear and apple had little or no activity. Tomato extract (20-50 microl of 100% juice) inhibited both ADP- and collagen-induced aggregation by up to 70% but could not inhibit arachidonic acid-induced platelet aggregation and concomitant thromboxane synthesis under similar experimental conditions. The anti-platelet components (MW <1000 Da) in tomatoes are water soluble, heat stable and are concentrated in the yellow fluid around the seeds. The active fractions were separated using gel filtration and HPLC. The aqueous fraction (110 000 xg supernatant) of tomatoes containing anti-platelet activity was subjected to gel filtration column chromatography (Biogel P2 column). The activity was fractionated into two peaks, peak-3 and peak-4 (major peak). Subsequently, peak-4 was further purified by HPLC using a reversed-phase column. NMR and mass spectroscopy studies indicated that peak F2 (obtained from peak 4) contained adenosine and cytidine. Deamination of peak F2 with adenosine deaminase almost completely abolished its anti-platelet activity, confirming the presence of adenosine in this fraction. In comparison, deamination of peak-4 resulted in only partial loss of inhibitory activity while the activity of peak-3 remained unaffected. These results indicate that tomatoes contain anti-platelet compounds in addition to adenosine. Unlike aspirin, the tomato-derived compounds inhibit thrombin-induced platelet aggregation. All these data indicate that tomato contains very potent anti-platelet components, and consuming tomatoes might be beneficial both as a preventive and therapeutic regime for cardiovascular disease.

Vitamin E requirements, transport, and metabolism : role of α-tocopherol-binding proteins

Abstract Vitamin E (RRR-α-tocopherol) is a lipid-soluble antioxidant that is present in the membranes of intracellular organelles. There it plays an important role in the suppression of free radical-induced lipid peroxidation. There are eight naturally occurring homologues of vitamin E that differ in their structure and in biological activity in vivo and in vitro. Although γ-tocopherol is a more effective free radical scavenger than α-tocopherol in vitro, the reverse is true in vivo, suggesting that the tocopherol distribution systems favor the localization of α-tocopherol at the sites where it is required. Vitamin E is transported in plasma primarily by lipoproteins, but little is known of how it is transported intracellularly. A 30 kDa α-tocopherol-binding protein in the liver cytoplasm may regulate plasma vitamin E concentrations by preferentially incorporating the vitamin E homologue, RRR-α-tocopherol (α-tocopherol), into nascent very low density lipoproteins. However, this α-tocopherol-binding protein is unique to the hepatocyte, whereas α-tocopherol is present in the cells of all major tissues. Moreover α-tocopherol accumulates at those sites within the cell where oxygen radical production is greatest and thus where it is most required; in the membranes of heavy mitochondria, light mitochondria, and endoplasmic reticulum. This raises the question of how the lipid-soluble α-tocopherol is transported intracellularly in different tissues. We have identified a new α-tocopherol-binding protein of molecular mass 14.2 kDa in the cytosol of heart and liver. This protein specifically binds α-tocopherol in preference to the δ- and γ-homologues but does not bind oleate. Studies on immunoreactivity and ligand specificity of the protein suggest that it is not a fatty acid-binding protein. The 14.2 kDa α-tocopherol-binding protein stimulates the transfer of α-tocopherol from liposomes to mitochondria in vitro by 8 to 10 fold. We suggest that this low molecular mass TBP may be responsible for the intracellular transport and distribution of α-tocopherol in the tissues.

Dietary components and human platelet activity

Platelet hyperactivity is one of the most important factors responsible for the incidence of cardiovascular disease. There are many nutritive and non-nutritive compounds present in the diet which may affect platelet function in various ways. Recent discovery of anti-platelet factors in plants, vegetables and fruits provides a new dietary means for a long-term strategy to favorably modify human blood platelet activity. This review summarises the effects of these dietary components on human platelet function both in vitro and in vivo .

Plasma membrane fatty acid-binding protein (FABPpm) is exclusively located in the maternal facing membranes of the human placenta

We reported earlier the presence of a 40 kDa plasma membrane fatty acid‐binding protein (FABPpm) in human placenta. This protein is thought to be involved in the sequestration of unesterified free fatty acids bound to albumin from the maternal plasma for delivery to the fetus. However, its location in human placental syncytiotrophoblasts is not known. These cells are bipolar; one side facing maternal circulation (microvillous membranes), and the other side facing fetal circulation (basal membranes). Therefore, it is important to resolve the location of this protein in trophoblast membranes in order to understand fatty acid transport and metabolism in human placenta. Isolated plasma membranes vesicles were prepared respectively from the maternal facing microvillous and fetal facing surface of the human full‐term placental syncytiotrophoblast. Using these membrane preparations, fatty acid binding activity, the polyacrylamide gel electrophoresis radiobinding assay for FABPpm, and Western blot analysis of FABPpm were carried out to determine the location of this protein in these membranes. Based on the above studies we conclude that the FABPpm is located exclusively in the microvillous membranes. Since FABPpm may be responsible for FFA uptake, its location in the microvillous membranes favours the unidirectional flow of maternal FFA to the fetus.