Effect of diets rich in oleic acid, stearic acid and linoleic acid on postprandial haemostatic factors in young healthy men.

The aim of the present study was to investigate the effects of stearic acid-, oleic acid- and linoleic acid-rich meals on postprandial haemostasis in young healthy volunteers whose background diets had been controlled for 14 d in a residential study. Six healthy male volunteers were assigned randomly to consume diets rich in stearic acid, oleic acid or linoleic acid for 14 d. On day 15, plasma lipids and haematological variables were measured in the fasted state, and 3 and 7 h (factor VII and prothrombin activation peptide fragments, 1 and 2 only) after consumption of a test meal. Test meals provided 40 % of the subjects' daily energy requirement, with 41 % of the energy provided as fat, 17 % energy as protein and 42 % energy as carbohydrate. The mean fat content of the meal was 45 (sd 5) g. Significant alterations from fasted values were observed for activated factor VII after 7 h), factor VII antigen after 7 h), prothrombin activation peptide fragments 1 and 2 after 7 h) and plasminogen activator inhibitor type 1 activity after 3 h) after consumption of each of the three meals. No significant differences were observed in haemostatic values (factor VII coagulant activity, factor VII antigen, tissue plasminogen activator activity prothrombin activation peptide fragment and plasminogen activator inhibitor type-1) with regard to diet except for activated factor VII at 3 h; values were higher after the oleic acid- and linoleic acid-rich meals than after the stearic acid-rich meal After consumption of each of the three meals, chylomicrons contained proportionately more palmitic acid than the lipids ingested. The present study shows that there are demonstrable changes in postprandial haemostasis when young healthy volunteers with controlled dietary backgrounds are challenged with a physiological fat load. These changes are independent of the fatty acid composition of the test meals.

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 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.

Cellular uptake of long-chain fatty acids: role of membrane-associated fatty-acid-binding/transport proteins.

The critical importance of long-chain fatty acids in cellular homeostasis demands an efficient uptake system for these fatty acids and their metabolism in tissues. Increasing evidence suggests that the plasma-membrane-associated and cytoplasmic fatty-acid-binding proteins are involved in cellular fatty acid uptake, transport and metabolism in tissues. These binding proteins may also function in the fine tuning of cellular events by modulating the metabolism of long-chain fatty acids implicated in the regulation of cell growth and various cellular functions. Several membrane-associated fatty-acid-binding/transport proteins such as plasma membrane fatty-acid-binding protein (FABPpm, 43 kDa), fatty acid translocase (FAT, 88 kDa) and fatty acid transporter protein (FATP, 63 kDa) have been identified. In the feto-placental unit, preferential transport of maternal plasma arachidonic and docosahexaenoic acids across the placenta is of critical importance for fetal growth and development. Our studies have shown that arachidonic and docosahexaenoic acids are preferentially taken up by placental trophoblasts for fetal transport. The existence of a fatty-acid-transport system comprising multiple membrane-binding proteins (FAT, FATP and FABPpm) in human placenta may be essential to facilitate the preferential transport of maternal plasma fatty acids in order to meet the requirements of the growing fetus. The preferential uptake of arachidonic and docosahexaenoic acids by the human placenta has the net effect of shunting these maternal plasma fatty acids towards the fetus. The roles of plasma membrane-associated binding/transport proteins (FABPpm, FAT and FATP) in tissue-specific fatty acid uptake and metabolism are discussed.

Further characterization of a novel triacylglycerol hydrolase activity (pH 6.0 optimum) from microvillous membranes from human term placenta.

We recently identified the presence of two distinct triacylglycerol hydrolases with pH optima of 6.0 and 8.0 in human placental microvillous membranes (MVM). The TAG hydrolase with a pH optimum of 8.0 has properties similar to lipoprotein lipase, whereas TAG hydrolase with a pH optimum of 6.0 still to be fully characterized. In order to understand the functional and structural relationships between these two TAG hydrolases of MVM we have further investigated their biochemical and molecular properties. The presence of oleic acid inhibited TAG hydrolase activity with a pH optimum of 8.0 by 60 per cent whilst it had very little effect on the pH 6.0 TAG hydrolase activity. K(m)values for TAG hydrolases at pH 6.0 and pH 8. 0 optima were 170.6 and 9.83 nmol triolein, respectively, whereas the corresponding V(max)values were 0.32 and 0.037 nmol oleic acid/min mg/protein. Treatment of MVM with phenylmethylsulphonofluoride or protamine had no effect on TAG hydrolase at pH 6.0 whereas both decreased activity at pH 8.0, by 70 per cent and 52 per cent, respectively (P< 0.05), compared with control. p-Chloromercuribenzoate inhibited both TAG hydrolase activities by 25-30 per cent whereas iodoacetate inhibited TAG hydrolase activity with optimum pH 8.0 by 74 per cent and the activity at pH 6.0 by 28 per cent. Unlike the TAG hydrolase activity at pH 8.0, the activity at pH 6.0 was not affected by heparin. TAG hydrolase activity at pH 6.0 was significantly decreased compared with that of pH 8.0 optimum TAG hydrolase activity in smokers placenta. A threefold increase in pH 6.0 TAG hydrolase activity was observed following differentiation, whereas membrane associated TAG hydrolase activity with optimum pH 8.0 did not change. The TAG hydrolase with optimum pH 6.0 was subsequently purified from MVM to almost 1000-fold enrichment of the activity over the starting material. The final preparation however, still contained three distinct protein bands (90, 70 and 45 kDa). When extracted from non-denaturing polyacrylamide gels, the 70 kDa protein was the only protein to have TAG hydrolysing activity and had a pH optimum of 6.0. Labelling of samples with [(14)C]tetrahydrolipstatin also confirmed that the TAG hydrolase active protein was a 70 kDa protein. In conclusion, we report that there is a 70 kDa TAG hydrolase with optimum pH 6.0 in human placental MVM which is quite distinct from placental lipoprotein lipase.

A residential study comparing the effects of diets rich in stearic acid, oleic acid, and linoleic acid on fasting blood lipids, hemostatic variables and platelets in young healthy men.

Dietary fat is known to influence the variables of blood coagulation and fibrinolysis associated with vascular disease. However, the role of fat content and/or fat composition of the diet in this regard is still not well understood. In the present study, we investigated the effects of three isoenergic diets of differing fat composition in nine healthy young men in a strictly controlled residential study. Subjects consumed the three experimental diets for periods of 2 weeks each, separated by a washout period of at least 5 weeks in a randomized crossover design. The diets provided 38% of total energy intake as fat, 45% as carbohydrate, and 17% as protein, and differed only with respect to the fatty acid composition (stearic acid-rich diet: 34.1% stearic acid, 36.6% oleic acid; oleic acid-rich diet: 65.8% oleic acid; linoleic acid-rich diet: 36.5% linoleic acid, 38% oleic acid). Blood samples were collected at the beginning and at the end of each dietary period from fasted subjects for determination of factor VII coagulant activity (FVIIc), activated factor VII (FVIIa), factor VII antigen (FVIIag), tissue plasminogen activator (tPA) activity, plasminogen activator inhibitor type 1 (PAI-1) activity, fibrinogen, prothrombin fragment 1+2 (F(1+2)), and plasma lipids. There were no significant differences between diets in fasting plasma concentrations of FVIIc, FVIIa, FVIIag, fibrinogen, F(1+2), PAI-1 activity, and tPA activity. Plasma concentrations of lipids (high density lipoproteins, low density lipoproteins, triacylglycerols, and total cholesterol) were also unaffected. Although there were no changes in platelet aggregation response and membrane fluidity observed in any of the diets, increased anti-aggregatory prostaglandin E(1) binding to platelet membranes was observed only in the case of linoleic acid-rich diet. In conclusion, diets with very different fatty acid compositions, at 38% of energy as fat intake, did not significantly influence blood coagulation, fibrinolysis, or blood lipids in the fasting state in young healthy men.

Preferential distribution of long chain polyunsaturated fatty acids in phospatidyl ethanolamine fraction of guinea pig alveolar apical membranes.

We investigated the fatty acid distribution in guinea pig alveolar apical membranes at different developmental stages. Fatty acid composition of the purified membranes isolated from guinea pig fetuses (at 65 day, term=68 day), neonates (day 1) and adult males was determined. The levels of arachidonic acid (AA) and docosahexaenoic acid (DHA) were higher in the adult guinea pig alveolar apical membrane phosphatidylethanolamine (PE) fraction (9. 3+/-2.2 and 2.9+/-1.0%, respectively) while in other phospholipids (PL) fractions their levels were low or absent (P<0.01). Furthermore, levels of AA and DHA in the PE fraction of apical membrane increased significantly from fetal (6.6+/-3.0 and 0.8+/-0.4%, respectively) to neonatal life (10.3+/-1.5 and 3.0+/-0.8%, respectively). Increase in the level of DHA (almost four-fold) was much more pronounced than that of AA (P<0.05). As for guinea pig alveolar membranes, EPA and AA were mostly present in the PE fraction in pulmonary adenocarcinoma derived cells (A549 cells), a parallel model of type II pneumocytes, with the levels of AA around three-fold greater than that of EPA, Binding of radiolabelled fatty acids to A549 cells showed no significant differences between the maximum uptake achieved for different fatty acids (AA, 1.7+/-0.2, EPA, 2.3+/-0.3, LA, 1.7+/-0.2, OA, 2.0+/-0.2nmol/mg protein, P>0.5). Once the fatty acids were taken up by these cells AA was mostly identifiable in the monoacylglycerol (MAG) fraction, whereas EPA was equally distributed between the MAG and PL fractions. Oleic acid was mainly present in the triglyceride (TAG) fraction whereas LA was evenly distributed between the TAG, MAG, and PL fractions. Our data demonstrate a preferential distribution of AA and DHA in PE fractions of alveolar apical membranes during development.

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).

Molecular mechanism of cellular uptake and intracellular translocation of alpha-tocopherol: role of tocopherol-binding proteins.

Vitamin E (alpha-tocopherol) is a lipid-soluble antioxidant which is present in cellular membranes where it plays an important role in the suppression of free radical-induced lipid peroxidation. There are eight naturally occurring homologues of vitamin E which differ in their structure and in biological activity in vivo and in vitro. Various studies have suggested that the tocopherol distribution system favours the accumulation of alpha-tocopherol both in the plasma and different tissues. Mechanisms involved in the preferential accumulation of alpha-tocopherol are not yet well established; however, recent data indicate that both intracellular and membrane alpha-tocopherol-binding proteins may be involved in these processes. A 30 kDa alpha-tocopherol-binding protein (TBP) in the liver cytoplasm is now known to regulate plasma vitamin E concentrations by preferentially incorporating alpha-tocopherol into nascent very low density (VLDL) whereas the 15 kDa TBP may be responsible for intracellular distribution of alpha-tocopherol. The 30 kDa TBP is unique to the hepatocyte whereas the 15 kDa TBP is present in all major tissues. The 15 kDa TBP specifically binds alpha-tocopherol in preference to the delta- and gamma-tocopherol and may exclusively transport alpha-tocopherol to these intracellular sites. In addition, the presence of a membrane TBP (TBPpm) in tissues may regulate their alpha-tocopherol levels. Activity of erythrocyte TBPpm appears to be reduced in smokers, which may lead to reduced levels of alpha-tocopherol in these cells despite smokers have similar plasma levels of vitamin E as in non-smokers. The current status of the evidence for this directed flow of alpha-tocopherol through interactions with these proteins (TBP and TBPpm) is discussed.

The effects of structurally defined triglycerides of differing fatty acid composition on postprandial haemostasis in young, healthy men.

The aim of this study was to investigate whether a number of key haemostatic factors were altered when healthy young individuals were challenged with a fat load of physiological size contained within a meal composed of normal ingredients and whether this response was modified when the fatty acid composition of the meal was altered radically. Eight healthy male volunteers each randomly consumed four meals which were identical in terms of gross nutritional content (41% of energy provided as fat, 17% as protein and 42% as carbohydrate) but which differed in fatty acid composition. To reduce the possible influence of fatty acid position within the triglyceride molecule on lipid absorption and subsequent metabolic effects, the structural integrity of 91% of fat (test triglycerides such as 1,3 distearoyl-2-oleoyl glycerol (S-O-S), trioleine (O-O-O), and 1,3 dilinoleoyl-2-oleoyl glycerol, (L-O-L)) in the meals was controlled so that the principal fatty acid in the sn-2 position was oleic acid (18:1n-9). Meals rich in either a test triglyceride or a control oil provided 44+/-6 g of fat. No significant alterations from fasted values of elevated plasma factor VII coagulant activity (FVIIc) or F1 + 2 were observed. FVIIA varied significantly over the postprandial time course; however, when expressed as a percentage of the fasting value, the FVIIa responses to O-O-O and L-O-L differed significantly but this was not evident when the absolute values were analysed. Similarly, no difference in plasma fibrinopeptide A (FPA) concentrations were evident. After all four meals, chylomicron contained proportionately more palmitic acid and generally less oleic acid than the ingested lipids. This study clearly demonstrates that postprandial haemostatic responses of young healthy individuals to a physiological fat load are minimal, (irrespective of triglyceride structure).

Inhibitory effect of Ginkgo biloba extract on human platelet aggregation.

The effect of pure flavonoids and Gingko biloba extract (GBE) on human platelet aggregation was investigated. Most of the flavonoids and vitamin E did not affect platelet aggregation in platelet-rich plasma (PRP); however some of these flavonoids inhibited platelet aggregation in gel-filtered platelets (GFP). GBE inhibited both ADP- and collagen-induced platelet aggregation in PRP, GFP and in whole blood in a dose-dependent manner. GBE at very low concentrations inhibited whole blood aggregation induced by ADP compared with those used for PRP or GFP. Flavonoids and GBE decreased the production of TxA(2) induced by collagen and ADP in PRP. However, no correlation was observed between the inhibition of platelet aggregation and the decrease of TxA(2) synthesis. GBE and flavonoids did not affect platelet membrane fluidity. However, the incubation of PRP with GBE increased cAMP levels in platelets, which is known to inhibit platelet activation by lowering intracellular Ca2+ levels. GBE is a mixture of many compounds, including flavonoids and gingkoglides, which affect metabolism of cAMP, TxA(2) and Ca2+ in platelets. It is effective in the inhibition of platelet aggregation, both in PRP and whole blood, and thus may be potentially used as an effective oral anti-platelet therapeutic agent.

Characterisation of triacylglycerol hydrolase activities in human placenta.

Triacylglycerol hydrolase activities were characterised in homogenates, cytosol, and microvillous membranes (MVM) of human placenta. Homogenates of placenta exhibited three distinct triacylyglycerol hydrolase activities with pH optima 4.5, 6.0 and 8. 0. On further fractionation, placental cytosol exhibited both acid cholesterol ester hydrolase (pH 4.5) and hormone sensitive lipase (pH 6.0) activities, whereas purified placental MVM exhibited two distinct triacylyglycerol hydrolase activities; a minor activity at pH 8.0 and a second major activity at pH 6.0. Triacylglycerol hydrolase activity at pH 8.0 of MVM appeared to be lipoprotein lipase (consistent with criteria such as serum stimulation and salt inhibition), whereas at pH 6.0 the activity was unique in that it was almost abolished by serum, but was not affected by high NaCl concentrations. Our data, for the first time, demonstrate that human placental MVM, in addition to lipoprotein lipase, contain a newly identified triacylglycerol hydrolase activity at pH 6.0.

Differential distribution and metabolism of arachidonic acid and docosahexaenoic acid by human placental choriocarcinoma (BeWo) cells.

The time course of incorporation of [14C]arachidonic acid and [3H]docosahexaenoic acid into various lipid fractions in placental choriocarcinoma (BeWo) cells was investigated. BeWo cells were found to rapidly incorporate exogenous [14C]arachidonic acid and [3H] docosahexaenoic acid into the total cellular lipid pool. The extent of docosahexaenoic acid esterification was more rapid than for arachidonic acid, although this difference abated with time to leave only a small percentage of the fatty acids in their unesterified form. Furthermore, uptake was found to be saturable. In the cellular lipids these fatty acids were mainly esterified into the phospholipid (PL) and the triacyglycerol (TAG) fractions. Smaller amounts were also detected in the diacylglycerol and cholesterol ester fractions. Almost 60% of the total amount of [3H]Docosahexaenoic acid taken up by the cells was esterified into TAG whereas 37% was in PL fractions. For arachidonic acid the reverse was true, 60% of the total uptake was incorporated into PL fractions whereas less than 35% was in TAG. Marked differences were also found in the distribution of the fatty acids into individual phospholipid classes. The higher incorporation of docosahexaenoic acid and arachidonic acid was found in PC and PE, respectively. The greater cellular uptake of docosahexaenoic acid and its preferential incorporation in TAG suggests that both uptake and transport modes of this fatty acid by the placenta to fetus is different from that of arachidonic acid.

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.

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.

Interaction of free fatty acids with human leptin.

Relatively high concentrations of leptin are present in plasma and it is thought to play a major role in lipid homeostasis. Leptin is reported to lower tissue triglyceride content by increasing intracellular oxidation of free fatty acids (FFA). However very little is known regarding the interaction between leptin and plasma FFA. We studied the interaction of FFA with leptin using a direct radiolabelled fatty acid binding assay, a fluorescence assay, electrophoretic mobility and autoradiobinding. All these data indicate that binding of FFA with leptin is reversible and shows a positive co-operativity. The binding of FFA to leptin produces a change in the pI value of the leptin and also increased the electrophoretic mobility of the protein in native polyacrylamide gels. The change in leptin's electrophoretic mobility depends on the chain length and the number of double bonds of the fatty acid, as stearic acid, 18:0, had no effect whereas oleic acid, 18:1n-9, linoleic acid, 18:2n-6, arachidonic acid, 20:4n-6, and docosahexaneoic acid, 22:6n-3, affected leptin's mobility to different degrees. The physiological implication of leptin-FFA interaction is not known, however the interaction may depend on the plasma FFA composition and concentration which are known to vary in different pathological/physiological conditions.

G protein-coupled prostaglandin receptor modulates conductive Na+ uptake in lung apical membrane vesicles.

Because G protein-regulated cation channels in type II pneumocytes constitute the most likely pathway for alveolar Na+ entry, we explored the hypothesis that a G protein-coupled prostaglandin (PG) E2 receptor controls perinatal lung alveolar Na+ transport. [3H]PGE2 binding to the alveolar apical membrane was trypsin sensitive and showed a rank order of competitive inhibition: PGE2 = PGE1 > PGD2 > PGF2 alpha. Kinetic analysis demonstrated both high-affinity [dissociation constant (KD) = 2.1 +/- 0.7 nM; maximal binding (Bmax) = 27 +/- 7 fmol/mg protein] and low-affinity (KD = 28 +/- 2 nM; Bmax = 265 +/- 29 fmol/mg protein) binding sites. Modulation of high-affinity GTPase activity identified a similar potency order (IC50 = 11 mM for PGF2 alpha vs. 10-50 microM for other PGs), suggesting that the receptors are G protein coupled. Finally, 1 microM PGE2 (approximately IC25) increased conductive 22Na+ uptake into membrane vesicles only in the presence of 100 microM intravesicular GTP. The KD value for the high-affinity binding site together with the rank order of PG effect on ligand binding and G protein function places this PG receptor in the EP3 subtype, whereas Na+ uptake studies suggest that it helps maintain perinatal lung Na+ homeostasis.

alpha-Tocopherol binding activity of red blood cells in smokers.

Despite high plasma levels of vitamin E, red blood cell membranes contain relatively low levels of vitamin E. This suggests the existence of a selective vitamin E uptake/regeneration system in human red blood cell membranes. alpha-Tocopherol binding sites on human red blood cells are thought to be involved in the uptake of alpha-tocopherol from the plasma. To understand the role of the uptake system we have compared the alpha-tocopherol content and binding activity of red blood cells from smokers and non-smokers. The specific binding of [3H] alpha-tocopherol to pure red blood cell preparations from smokers (n = 7, 28.4 +/- 2.8 years) was 30.6 +/- 3.2 fmoles per 3 x 10(8) red blood cells and for non-smokers (n = 17, 27.9 +/- 1.3 years) was 41.7 +/- 3.7 fmoles per 3 x 10(8) red blood cells. Thus alpha-tocopherol uptake activity was significantly lower in smokers (P = 0.05). Red blood cells from smokers contained less (1.8 +/- 0.4 micrograms/gHb) alpha-tocopherol than non-smokers (2.8 +/- 0.3 micrograms/gHb), (P < 0.05), despite plasma levels of alpha-tocopherol being similar: 12.9 +/- 0.8 microM in non-smokers vs. 12.7 +/- 0.5 microM in smokers. However, adjusting plasma alpha-tocopherol for total plasma cholesterol plus triacylglycerols showed alpha-tocopherol levels were higher (P < 0.01) in non-smokers (2.84 +/- 0.10 mumol alpha-tocopherol/ mmol [cholesterol+triacylglycerol]) than in smokers (2.36 +/- 0.11 mumol alpha-tocopherol/mmol [cholesterol+triacylglycerol]). The reduced alpha-tocopherol levels in red blood cells from smokers may be due to impairment of alpha-tocopherol uptake activity. The reduced levels of alpha-tocopherol in smokers red blood cells was not associated with any changes in cell membrane fluidity. At present it is not known whether supplementation of smokers with vitamin E would normalise the alpha-tocopherol uptake activity of red blood cells.

High D-glucose does not affect binding of alpha-tocopherol to human erythrocytes.

The role of alpha-tocopherol uptake system in human erythrocyte in the uptake of plasma alpha-tocopherol has been suggested. However no information is available on alpha-tocopherol uptake activity of human erythrocytes in the presence of high levels of D-glucose which is known to lead to pathological alterations in different cells including human erythrocytes. Therefore, in order to examine the effect of D-glucose on the binding of alpha-tocopherol to human erythrocytes, the binding characteristics of alpha-tocopherol to these cells were established first. Binding of [3H]alpha-tocopherol to human erythrocytes was both saturable and specific. Scatchard analysis of alpha-tocopherol binding to these cells showed the presence of two independent classes of binding sites with widely different affinities. The high affinity binding sites had a dissociation constant (Kd1) of 90 nM with a binding capacity (n1) of 900 sites per cell, whereas the low affinity binding sites had a dissociation constant (Kd2) of 5.2 microM and a binding capacity (n2) of 105,400 sites per cell. Trypsin treatment abolished all the alpha-tocopherol binding activity. Competition for the binding of alpha-tocopherol to human erythrocytes was effective with other homologues of alpha-tocopherol (beta-tocopherol, gamma-tocopherol and delta-tocopherol) and their potency was almost equal to alpha-tocopherol itself. The order of preference was alpha-tocopherol > beta-tocopherol > or = gamma-tocopherol > or = delta-tocopherol. Incubation of human erythrocytes with various concentrations of D-glucose did not affect alpha-tocopherol uptake activity. Our data demonstrate the presence of an alpha-tocopherol uptake system in human erythrocytes and that the alpha-tocopherol uptake activity is not modulated by the presence of D-glucose.