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Biomedical subjects

A K Dutta-Roy

Publications and source records attributed to A K Dutta-Roy.

At least 19 recordsLinked to original sources
PubMed1999

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).

Adult↗
PubMed1998

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.

Blood↗
PubMed1998

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.

Carrier Proteins↗
PubMed1998

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.

Arachidonic Acids↗
PubMed1998

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.

Arachidonic Acid↗
PubMed1998

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.

Animals↗
PubMed1997

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.

Binding Sites↗
PubMed1997

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.

Adult↗
PubMed1997

Uptake of long chain fatty acids by human placental choriocarcinoma (BeWo) cells: role of plasma membrane fatty acid-binding protein.

In order to understand the mechanisms by which fatty acids are taken up by the placenta, the uptake of oleic, linoleic, arachidonic, and docosahexaenoic acids by cultured human placental choriocarcinoma (BeWo) cells was examined. Fatty acid uptake by BeWo cells was temperature-dependent and exhibited saturable kinetics. Oleic acid was taken up least and docosahexaenoic acid most by these cells. Moreover, competitive studies of fatty acid uptake by BeWo cells also indicated preferential uptake compared with oleic acid in the order of docosahexaenoic acid, arachidonic acid, and linoleic acid. Western blot analysis demonstrated that BeWo cells express a protein immunoreactive with antibodies to the human placental plasma membrane fatty acid-binding protein (p-FABPpm). Furthermore, pre-treatment of BeWo cells with these antibodies inhibited most of the uptake of docosahexaenoic (64%) and arachidonic acids (68%) whereas oleic acid uptake was inhibited only 32% compared with the controls treated with preimmune serum. These results clearly demonstrate that the pFABPpm may be involved in the preferential uptake of essential fatty acids (EFA) and their long chain polyunsaturated fatty acids (LCPUFA) by these cells. Studies on the distribution of radiolabeled fatty acids in the cellular lipids of BeWo cells showed that docosahexaenoic acid was incorporated mainly in the triacylglycerol fraction, followed by the phospholipid fraction, whereas for arachidonic acid the reverse was true. The preferential incorporation of docosahexaenoic acid into triacylglycerol suggests that triacylglycerol may play an important role in the placental transport of docosahexaenoic acid to the fetal circulation. Together these results demonstrate the preferential uptake of EFA/LCPUFA by BeWo cells that is most probably mediated via the pFABPpm. We thus propose that the p-FABPpm may be involved in the sequestration of maternal plasma LCPUFA by the placenta.

Antibodies↗
PubMed1996

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 alpha-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 (approximately 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.

Arachidonic Acid↗