Lowering of platelet aggregation and serum eicosanoid levels in rats fed with a diet containing coconut oil blends with rice bran oil or sesame oil.

The present investigation was undertaken to study the effects of feeding a diet containing blended and interesterified fat to rats on thrombotic parameters such as platelet aggregation and eicosanoid levels in blood serum. Male Wistar rats were fed with a diet containing 10% fat from native; coconut oil (CNO), rice bran oil (RBO), sesame oil (SESO), blended; (CNO+RBO blend (B), CNO+SESO(B), or interesterified oils; CNO+RBO interesterified (I), CNO+SESO(I) for a period of 60 days. Rats given a diet containing blended oil of CNO+RBO(B) or CNO+SESO(B) showed a decrease in rate of ADP induced aggregation of platelets by 34% and 30%, respectively, compared to those fed with CNO. Aggregation induced by collagen was also reduced similarly in rats given blended or interesterified oils of CNO with RBO or SESO. Feeding interesterified oil CNO+RBO(I), and CNO+SESO(I) to rats also resulted in decrease in rate of ADP induced platelet aggregation by 37% and 34%, respectively, compared to rats fed with CNO. The prostacyclin/thromboxane ratio in serum was increased in rats fed with blended and interesterified oil compared to those fed with CNO. These results indicated that CNO when blended or interesterified with RBO or SESO exhibit antithrombotic effects as compared to the effect observed by feeding rats with CNO.

Micellar oleic and eicosapentaenoic acid but not linoleic acid influences the beta-carotene uptake and its cleavage into retinol in rats.

Improving the bioavailability of beta-carotene is vital to manage vitamin A deficiency. The influence of micellar oleic (OA), linoleic (LA) and eicosapentaenoic (EPA) acids on plasma beta-carotene response and its conversion to retinol has been studied in rats employing single (9 h time course) and repeated (10 days) dose administrations. After a single dose, the levels (area under the curve) of plasma beta-carotene and retinyl palmitate in OA and EPA groups were higher (p < 0.05) by 13, 7 and 11, 6 folds than LA group. The liver beta-carotene level in OA and EPA groups were higher (p < 0.05) by 3 and 1.2 folds than LA group. After repeated dose, the plasma beta-carotene and retinyl palmitate levels in OA (6.2%, 51.7%) and EPA (25.4%, 17.23%) groups were higher (p < 0.05) than LA group. The liver beta-carotene level in OA (21.2%) and EPA (17.6%) groups were higher (p < 0.05) than LA group. In both the experiments, the activity of beta-carotene 15,15'-dioxygenase in the intestinal mucosa and plasma triglyceride levels were also higher in OA and EPA groups than LA group. beta-Carotene excreted through urine and feces of OA and EPA groups was lower than the LA group. These results demonstrate an improved absorption and metabolism of beta-carotene when fed mixed micelles with OA or EPA compared with LA. Although the mechanism involved in selective absorption of fatty acids needs further studies, intestinal beta-carotene uptake and its conversion to vitamin A can be modulated using specific fatty acids.

Enhanced lutein bioavailability by lyso-phosphatidylcholine in rats.

The bioavailability of lutein solubilized in mixed micelles containing either phosphatidylcholine (PC) or lysophosphatidylcholine (lysoPC) was evaluated in male rats. Mixed micelles contained 2.5 mM monooleoylglycerol, 7.5 mM oleic acid, 12 mM sodium taurocholate and 200 microM lutein either with 3 mM PC or lysoPC. To study lutein bioavailability, single and repeated dose experiments were conducted. For single dose study, group of rats (n = 30/group) were fed single dose of lutein solubilized in lysoPC (LPC group), PC (PC group) and no phospholipids (NoPL group) in micellar form. Each group was further divided in to five sub-groups (n = 6/sub group) to measure lutein bioavailability over time up to 9 h. For repeated dose study, group of rats (n = 6/group) were fed daily for 10 days a dose of lutein in mixed micelles with NoPL, PC and LPC. A separate group (n = 6) not fed mixed micelles was considered as zero-time control. In both the experiments, mixed micelles (0.2 ml/rat) were fed to the rat by direct intubation to the stomach. Results of single dose studies showed that the mean lutein levels in the plasma and liver of the PC group was significantly lower (p < 0.05) than those of the other two groups. Moreover, the average lutein level in the plasma and liver was significantly (p < 0.05) different among the groups in the order LPC > NoPL > PC. But, repeated dose experiment followed the order LPC > PC > NoPL. The level of lutein excreted through urine and feces of PC group was significantly higher (p < 0.05) than those of the other two groups. Thus, the results indicate that the PC in the mixed micelles suppressed the intestinal uptake of lutein after single dose but not after repeated dose and that lysoPC enhanced the absorption. In both the experiments, plasma and liver level of lutein was higher in LPC compared with PC group. Results also suggest that the luminal hydrolysis of PC to lysoPC is necessary for intestinal uptake of lutein solubilized in mixed micelles.

Modulation of antioxidant enzyme activities, platelet aggregation and serum prostaglandins in rats fed spray-dried milk containing n-3 fatty acid.

Spray-dried milk enriched with n-3 fatty acids from linseed oil or fish oil were fed to rats to study its influence on liver lipid peroxides, hepatic antioxidant enzyme activities, serum prostaglandins and platelet aggregation. Significant level of alpha linolenic acid, eicosapentaenoic acid and docosahexaenoic acid were accumulated at the expense of arachidonic acid in the liver of rats fed n-3 fatty acid enriched formulation. The linseed oil and fish oil enriched formulation fed group had 44 and 112% higher level of lipid peroxides in liver homogenate compared to control rats fed groundnut oil enriched formulation. Catalase activity in liver homogenate was increased by 37 and 183% respectively in linseed oil and fish oil formulation fed rats. The glutathione peroxidase activity decreased to an extent of 25-36% and glutathione transferase activity increased to an extent of 34-39% in rats fed n-3 fatty acids enriched formulation. Feeding n-3 fatty acid enriched formulation significantly elevated the n-3 fatty acids in platelets and increased the lipid peroxide level to an extent of 4.2-4.5 fold compared to control. The serum thromboxane B2 level was decreased by 35 and 42% respectively in linseed oil and fish oil enriched formulation fed rats, whereas, 6-keto- prostaglandin F1alpha level was decreased by 17 and 23% respectively in linseed oil and fish oil enriched formulation fed rats. The extent and rate of platelet aggregation was decreased significantly in n-3 fatty acids enriched formulation fed rats. This indicated that n-3 fatty acids enriched formulation beneficially reduces platelet aggregation and also enhances the activities of hepatic antioxidant enzymes such as catalase and glutathione transferase.

Modulation of antioxidant enzyme activities, platelet aggregation and serum prostaglandins in rats fed spray-dried milk containing n-3 fatty acid.

Spray-dried milk enriched with n-3 fatty acids from linseed oil (LSO) or fish oil (FO) were fed to rats to study its influence on liver lipid peroxides, hepatic antioxidant enzyme activities, serum prostaglandins and platelet aggregation. Significant level of alpha linolenic acid, eicosapentaenoic acid and docosahexaenoic acid were accumulated at the expense of arachidonic acid in the liver of rats fed n-3 fatty acid enriched formulation. The linseed oil and fish oil enriched formulation fed group had 44 and 112% higher level of lipid peroxides in liver homogenate compared to control rats fed groundnut oil enriched formulation. Catalase activity in liver homogenate was increased by 37 and 183% respectively in linseed oil and fish oil formulation fed rats. The glutathione peroxidase activity decreased to an extent of 25-36% and glutathione transferase activity increased to an extent of 34-39% in rats fed n-3 fatty acids enriched formulation. Feeding n-3 fatty acid enriched formulation significantly elevated the n-3 fatty acids in platelets and increased the lipid peroxide level to an extent of 4.2 to 4.5-fold compared to control. The serum thromboxane B2 level was decreased by 35 and 42% respectively in linseed oil and fish oil enriched formulation fed rats, whereas 6-keto-prostaglandin F1alpha level was decreased by 17 and 23% respectively in linseed oil and fish oil enriched formulation fed rats. The extent and rate of platelet aggregation was decreased significantly in n-3 fatty acids enriched formulation fed rats. This indicated that n-3 fatty acids enriched formulation beneficially reduces platelet aggregation and also enhances the activities of hepatic antioxidant enzymes such as catalase and glutathione transferase.

Inhibition of human platelet aggregation and membrane lipid peroxidation by food spice, saffron.

The inhibitory activity of saffron extract was studied on human platelets. Platelet aggregation and lipid peroxidation were evaluated with platelet rich plasma (PRP) and platelet membranes respectively obtained from blood of healthy human volunteers. Human platelets were subjected to stimulation with a variety of agonists like ADP (61 microM), epinephrine (76 microM), collagen (11 microg/ml), calcium ionophore A 23187 (6 microM) and ristocetin (1.25 microg/ml) in the presence and absence of saffron extract with IC50 being 0.66, 0.35, 0.86 and 0.59 mg respectively and no inhibition with ristocetin. The inhibitory effect was dose dependent with concentrations varying between 0.16 to 0.80 mg and time dependent at IC50. A significant decrease was observed in malondialdehyde (MDA) formed, one of the end products of arachidonic acid metabolism and of serotonin released from dense granules of platelets at respective IC50. Lipid peroxidation in platelet membranes induced by iron-ascorbic acid system was inhibited by saffron extract significantly with IC50 of 0.33 mg. Hence, it may be said that aqueous extract of saffron may have component(s), which protect platelets from aggregation and lipid peroxidation.

Cardamom extract as inhibitor of human platelet aggregation.

The inhibitory activity of cardamom extract was studied on human platelets. Platelet aggregation and lipid peroxidation were evaluated with platelet rich plasma (PRP) and platelet membranes, respectively, obtained from blood of healthy volunteers. Human platelets were subjected to stimulation with a variety of agonists including ADP (2.5 mM), epinephrine (2.5 mM), collagen (10 mM), calcium ionophore A 23187 (6 microM) and ristocetin (1.25 microg/mL). The IC50 were 0.49, 0.21, 0.55 and 0.59 mg with ADP, epinephrine, collagen and calcium ionophore A 23187, respectively, and no inhibition with ristocetin. The inhibitory effect was dose dependent with concentrations varying between 0.14 and 0.70 mg and time dependent at IC50. Lipid peroxidation induced by iron--ascorbic acid system in platelet membranes was analysed with malondialdehyde (MDA) as an index. An increase in concentration of cardamom has decreased the MDA formation significantly. Hence, it may be said that aqueous extract of cardamom may have component(s), which protect platelets from aggregation and lipid peroxidation.

Purification of a Class B1 platelet aggregation inhibitor phospholipase A2 from Indian cobra (Naja Naja) venom.

A platelet aggregation inhibitor phospholipase A(2) (NND-IV-PLA(2)) was isolated from Naja naja (Eastern India) venom by a combination of cation and anion exchange chromatography. NND-IV-PLA(2) is the most catalytically active enzyme isolated from the Indian cobra venom. The acidic PLA(2) profile of Eastern regional Indian cobra venom is distinctly different from that of the western regional venom. However the acidic PLA(2)s from both the regions follow the pattern of increasing catalytic activity with increase in acidic nature of the PLA(2) isoform. NND-IV-PLA(2) is a Class B1 platelet aggregation inhibitor and inhibits platelet aggregation induced by ADP, collagen and epinephrine. Modification of active site histidine abolishes both catalytic activity and platelet aggregation inhibition activities while aristolochic acid, a phospholipase A(2) inhibitor has only partial effect on the two activities.

Effect of dietary ghee--the anhydrous milk fat on lymphocytes in rats.

Lymphocytes are important components of the immune system. Dietary lipids affect the functioning of the immune system. Changes in the lipid composition of the lymphocyte membrane is a case in point. Membrane structural changes are reflected in the altered function of the cell. Lymphocyte proliferation and lymphocyte rosetting are membrane associated phenomena. Ghee, is a clarified butter product, commonly used in the Indian diet. It is rich in saturated fatty acids and also contain oxysterols which are generated on prolonged heating of ghee. Male weanling rats were fed 2.5% (of the total fat levels) of fresh or thermally oxidized ghee for a period of 8 weeks. The control rats were fed groundnut oil. Lipid composition of lymphocytes in ghee fed rats showed changes. In vitro lipid peroxidation of lymphocyte membranes increased by 26% in oxidized ghee fed rats. Na+K+ ATPase activity was decreased in oxidized ghee fed rats (18%). Lymphocyte proliferation was reduced in ghee fed rats (32%), compared to the controls, irrespective of the mitogens used (Con-A or PHA), or the tissue (splenocytes or peripheral blood lymphocytes). Oxysterols present in oxidized ghee are the likely agents inhibiting lymphoproliferation. Rosetting of lymphocytes decreased in the fresh ghee fed rats by 16% and in oxidized ghee fed rats by 25%. Membrane fluidity declined in the oxidized ghee fed rats. It is concluded that feeding ghee results in decreased proliferation of lymphocytes. Also, feeding oxidised ghee results in decreased proliferation of lymphocytes through alterations in the structure of the lymphocyte membranes in the rat.

Membrane changes in rat erythrocyte ghosts on ghee feeding.

Alterations in membrane lipid composition is known to result in functional and structural changes in the membrane, and dietary lipids play an important role in this change. It was of interest to study the influence of ghee feeding to the rat on membrane structure and function. The activities of membrane bound enzymes Na+ K+ ATPase and Acetylcholinesterase were studied as an index of membrane changes. Male weanling rats were fed 2.5% fresh or thermally oxidized ghee in the diet for a period of 8 weeks. The control rats were fed groundnut oil. A decrease of 28% in the membrane fluidity of erythrocyte ghost membranes was observed in the oxidized ghee fed group at 37 degrees C, by fluorescence polarization measurements using 1,6-Diphenyl-1,3,5-hexatriene as a probe. The activities of Na+ K+ ATPase and Acetylcholinesterase showed an increase of 65 and 200% respectively after feeding oxidized ghee (2.5%). Also changes in Na+, K+ and ATP kinetics were observed in these rats. Increased membrane lipid peroxidation (80%) and C/PL ratio (11%) in the oxidized ghee fed group was observed. Marginal changes in the fatty acid composition were also seen. Further, an increase in the osmotic fragility of erythrocytes was observed in the oxidized ghee fed rats. It is inferred from these experiments that consumption of oxidized ghee with the diet affects the erythrocyte ghost membrane structure and function at 2.5% level, whereas consumption of fresh ghee has no effect on the erythrocyte membrane.

Influence of retinol deficiency and curcumin/turmeric feeding on tissue microsomal membrane lipid peroxidation and fatty acids in rats.

The effect of retinol deficiency and curcumin/turmeric on lipid peroxidation and fatty acid profile was studied in liver, kidney, spleen and brain microsomes of rats. Results revealed an increase in lipid peroxidation in retinol deficient liver by 32%, kidney 30%, spleen 24% and brain 43% compared to the controls. Feeding 0.1% curcumin or turmeric for three weeks in diet to retinol deficient rats reduced the lipid peroxidation respectively to 12.5 or 22.6%, in liver, 23.7 or 24.1% in kidney, 14.4 or 18.0% in spleen and 16.0 or 31.4% in brain. Retinol deficiency lead to a reduction in the essential fatty acids. In liver C18:1 showed a reduction by 45.6%, C18:2 by 31.6% and C20:4 by 22.8%. In kidney C18:1 was reduced by 33.6%, 18:2 by 24.6% and 20:4 by 13.7%. In spleen and brain C18:1 showed a reduction by 10.2% and 33.9%, C18:2 by 37.9% and 12.1% and C20:4 by 15.7% and 35.3% respectively. Curcumin and turmeric fed group showed a significant increase in the abnormally reduced fatty acid levels.

Scavenging of superoxide anions by spice principles.

Effect of spice principles on scavenging of superoxide anion has been investigated. The superoxide anions, as measured by nitrobluetetrazolium (NBT) reduction in xanthine-xanthine oxidase system, were inhibited by superoxide dismutase, spice principles eugenol (cloves) and cuminaldehyde (cumin), antioxidants, butylated hydroxy toluene and butylated hydroxyanisole in a dose-dependent manner. The K(i) values for the inhibition of NBT reduction by eugenol and cuminaldehyde were 64 microM and 120 microM respectively. Zingerone (ginger) and linalool (coriander) inhibited NBT reduction to a maximum of 23 and 28% respectively. However, piperine (black pepper) and turmeric extracts (aqueous and acid) failed to scavenge superoxide anions.

5'-Nucleotidase activity in retinol deficiency induced albino rats.

Retinol deficient rat liver, kidney and spleen showed a significant decrease in their enzyme activity (36% to 50%) compared to controls. The lectins could stimulate the enzyme activity in retinol deficient group by 57.6 to 92%, compared to controls (13.3% to 74%). Detergents increased the enzyme activity in retinol deficient tissue microsomes by 4.5%-80% in comparison to controls (10.3% to 119%). The results reveal alterations in membrane structure induced by retinol deficiency.

Microsomal alkaline phosphatase activity in retinol deficiency induced albino rats.

The investigations showed a significant decrease in the alkaline phosphatase activity of retinol deficient liver (48.6%), kidney (65.8%) and spleen (61.9%), as compared to the controls (100%). An increase in Vmax and Km by 12 to 51.5% and 90.4 to 189%, respectively, was observed in all the tissues in the retinol deficient group, as compared to the controls. Subsequent freezing and thawing reduced the activity of alkaline phosphatase by 22.5 to 35.8% in the experimental group; whereas the reduction in the control group ranged from 8.8 to 21.5%. In the presence of lectins and detergents the activity of alkaline phosphatase decreased in both the groups to different levels.

Tissue Na+K+ activated adenosine triphosphatase in retinol deficient albino rats.

The effect of retinol deficiency on Na+K+ ATPase was studied. Retinol deficient rat kidney, liver and spleen revealed a two to three-fold increase in ouabain sensitive Na+K+ ATPase activity in relation to controls. The activity in the controls could be stimulated more by concanavalin A than in the deficient group. The membrane-bound activity in the deficient group was more susceptible to freezing and thawing than the controls. Detergents could enhance the activity in both the groups to different levels. The enzyme revealed a high Km and Vmax for ATP and the cholesterol: phospholipid ratio was markedly decreased in the deficient group.

PAF-acether in AK-5 tumour cells.

PAF-Acether fraction derived from stimulated AK-5 tumour cells, aggregated human platelets. The platelet aggregating ability increased linearly with increasing concentration of the stimulant, calcium ionophore A23187, and reached a maximum at 6 microM in 25 minutes. This factor had biological and chemical properties identical to authentic PAF-acether. Our results demonstrate that, although PAF-acether is produced mainly from pro-inflammatory cells, it appears to be produced even in tumour cells.

Influence of oryzanol on platelet aggregation in rats.

Platelet aggregation was measured using platelet rich plasma prepared from rats fed oryzanol in the control diet and those fed oryzanol in a 1 per cent cholesterol diet (HCD). Oryzanol with the control diet did not alter platelet aggregation induced by ADP and collagen. On the other hand, oryzanol fed along with HCD significantly inhibited platelet aggregation induced by ADP and totally inhibited aggregation induced by collagen.

Human placental 5'-nucleotidase: purification and properties.

Subcellular fractionation of human term placenta showed that the highest relative specific activity of 5'-nucleotidase and alkaline phosphatase resided in the microsomal fraction; of the total 5'-nucleotidase activity present, 7 per cent was in the cytosol. 5'-Nucleotidase was reproducibly purified over 500-fold in 17 per cent yield from the insoluble component of homogenates of term placenta to give a single major glycoprotein with two minor inactive protein contaminants. Purified placental 5'-nucleotidase was free from non-specific or alkaline phosphatase, hydrolysed 12 to 22 mumol AMP/min/mg of protein at 30 degrees C, and was activated up to fivefold by Triton X-100. AMP, Km 5 to 7 microM, was the preferred substrate. The Arrhenius plot was biphasic, with activation energies of 21.7 and 49.7 kJ/mol above and below 36 degrees C, the region of the transition temperature. Nucleoside di- and triphosphates inhibited competitively; the most potent inhibitors were ADP and adenosine 5'-methylenediphosphonate, Ki slope 90 nm and 6 nm, respectively. Lectins inhibited the enzyme; concanavalin A caused time-dependent inactivation reversible by alpha-methyl-D-mannoside. EDTA inactivated the enzyme; partial reactivation was achieved with divalent cations. The pH optimum was 7.2 to 7.3; Mg2+ produced a second alkaline pH optimum. The properties of placental 5'-nucleotidase are those of an intrinsic membrane protein and, in general, resemble properties of the several 'ecto'-5'-nucleotidases which have been purified from other tissues, although certain differences in kinetic properties of the placental enzyme are apparent.