Activation of the NO-cGMP pathway is associated with myocardial protection against ischemia. During ischemia, function of this pathway is disturbed. Little is known about the effects of supplements such as Red Palm Oil (RPO) on the myocardial NO- cGMP- signalling pathway. RPO consists of saturated (SFAs), mono-unsaturated (MUFAs) and polyunsaturated (PUFAs) fatty acids and is an antioxidant rich in natural B-carotene and vitamin E (tocopherols and tocotrienols). This study determined whether dietary RPO-supplementation protects against the consequences of ischemia and identified a possible mechanism for this protection. Long-Evans rats were fed a control diet or control diet plus 7 g RPO per kg diet for six weeks. Hearts were excised and mounted on a working heart perfusion apparatus. Cardiac function was measured before and after hearts were subjected to 25 minutes of global ischemia. Left ventricular systolic (LVSP) and diastolic pressure (LVDP), coronary flow (CF), heart rate (HR) and aortic output (AO) were measured. To assess NO-cGMP pathway activity, hearts subjected to the same conditions, were freeze-clamped and analysed for tissue cAMP and cGMP levels using a RIA method. Furthermore, composition of myocardial phospholipid fatty acids by gas chromatography and blood samples were collected for serum lipid determinations. The percentage aortic output recovery of hearts supplemented with RPO was 72.9 +/-3.43% vs 55.4 +/-2.48% for controls (P< 0.05). Ten minutes into ischemia the cGMP levels of the RPO-supplementation group were significantly higher than the control group (26.5+/-2.78 pmol/g vs 10.1+/-1.78 pmol/g. Total myocardial PUFA content in hearts supplemented with RPO increased from 54.45+/-1.11% before ischemia to 59.03 +/- 0.30% after ischemia P<0.05). Results demonstrated that RPO-supplementation protected hearts against the consequences of ischemia/reperfusion injury. These findings suggest that dietary RPO protects via the NO-cGMP pathway and/or changes in PUFA composition during ischemia/reperfusion.
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Effects of tocotrienols on cell viability and apoptosis in normal murine liver cells (BNL CL.2) and liver cancer cells (BNL 1ME A.7R.1), in vitro
Har CH, Keong CK.
Asia Pac J Clin Nutr. 2005;14(4):374-80.
The effects of tocotrienols on murine liver cell viability and their apoptotic events were studied over a dose range of 0-32 microg mL(-1). Normal murine liver cells (BNL CL.2) and murine liver cancer cells (BNL 1ME A.7R.1) were treated with tocotrienols (T(3)), alpha tocopherol (alpha-T) and the chemo drug, Doxorubicin (Doxo, as a positive control). Cell viability assay showed that T(3) significantly (P < or = 0.05) lowered the percentage of BNL 1ME A.7R.1 cell viability in a dose-responsive manner (8-16 microg mL(-1)), whereas T did not show any significant (P>0.05) inhibition in cell viability with increasing treatment doses of 0-16 microg mL(-1). The IC(50) for tocotrienols were 9.8, 8.9, 8.1, 9.7, 8.1 and 9.3 microg mL(-1) at 12, 24, 36, 48, 60 and 72 hours respectively. Early apoptosis was detected 6 hours following T(3) treatment of BNL 1ME A.7R.1 liver cancer cells, using Annexin V-FITC fluorescence microscopy assay for apoptosis, but none were observed for the non-treated liver cancer cells at the average IC(50) of 8.98 microg mL(-1) tocotrienols for liver cancer cells. Several apoptotic bodies were detected in BNL 1ME A.7R.1 liver cancer cells at 6 hours post-treatment with tocotrienols (8.98 microg mL(-1)) using Acridine Orange/Propidium Iodide fluorescence assay. However, only a couple of apoptotic bodies were seen in the non-treated liver cancer cells and the BNL CL.2 normal liver cells. Some mitotic bodies were also observed in the T(3)-treated BNL 1ME A.7R.1 liver cancer cells but were not seen in the untreated BNL 1ME A.7R.1 cells and the BNL CL.2 liver cells. Following T(3)-treatment (8.98 microg mL(-1)) of the BNL 1ME A.7R.1 liver cancer cells, 24.62%, 25.53% and 44.90% of the cells showed elevated active caspase 3 activity at 9, 12 and 24 hours treatment period, respectively. DNA laddering studies indicated DNA fragmentation occurred in the T(3)-treated liver cancer cells, BNL 1ME A.7R.1 but not in non-treated liver cancer cells and the T(3)-treated and non-treated normal liver cells. These results suggest that tocotrienols were able to reduce the cell viability in the murine liver cancer cells at a dose of 8-32 microg mL(-1) and that this decrease in percentage cell viability may be due to apoptosis.
The natural vitamin E tocotrienol (TCT) possesses biological properties not shared by tocopherols (TCP). Nanomolar alpha-TCT, not alpha-TCP, is potently neuroprotective (JBC 275:13049; 278:43508). Tocopherol-transport protein (TTP) represents the primary mechanism for maintaining normal alpha-TCP concentrations in plasma and extrahepatic tissues. TTP primarily transports alpha-TCP and has low affinity for alpha-TCT. There are no studies that have investigated tissue delivery of alpha-TCT when orally gavaged on a long-term basis. A long-term study was conducted to examine the effects of alpha-TCT or alpha-TCP supplementation, either alone or in combination, on tissue levels. Rats were maintained on a vitamin E-deficient diet and gavaged with alpha-TCT or alpha-TCP alone or in combination. Five generations of rats were studied over 60 weeks. TTP-deficient mice were supplemented with TCT and bred to examine tissue delivery of oral alpha-TCT. Orally supplemented alpha-TCT was effectively delivered to most tissues over time. When co-supplemented, alpha-TCP outcompeted alpha-TCT for transport systems delivering vitamin E to tissues. To evaluate the significance of TTP in alpha-TCT delivery to tissues, tissue levels of alpha-TCT in supplemented TTP-deficient mice were studied. alpha-TCT was transported to several vital organs in TTP-deficient mice. alpha-TCT restored fertility in TTP-deficient mice. In sum, orally supplemented alpha-TCT was successfully delivered to several vital organs. The transport efficiency of alpha-TCT to tissues may be maximized by eliminating the co-presence of alpha-TCP in the oral supplement. Examination of whether alpha-TCT may benefit humans suffering from neurological disorders because of congenital TTP deficiency is warranted.
A high performance liquid chromatographic (HPLC) method for the determination of tocopherols and tocotrienols in walnut samples is described. The compounds were extracted with n-hexane, using a simple solid-liquid extraction procedure. Tocol was used as internal standard and BHT as anti-oxidant. The ehromatographic separation was achieved using an Inertsil 5 SI normal phase column operating with isocratic elution of n-hexane/1,4-dioxane (96.5:3.5, v/v), at a flow rate of 0.7 mL/min. The effluent was monitored by a series arrangement of a diodearray detector followed by a fluorescence detector. The detection limits were low, between 0.037 and 0.266 (jig/mL. The method was precise (% CV less than 2.8%), accurate (% CV less than 5.6%), and, as a general rule, the recovery values were high (mean values ranging from 93.4% to 104.0%)
A comparison between tocopherol and tocotrienol effects on gastric parameters in rats exposed to stress
Azlina MF, Nafeeza MI, Khalid BA.
Asia Pac J Clin Nutr. 2005;14(4):358-65.
Rats exposed to stress developed various changes in the gastrointestinal tract and hormones. The present study was designed to compare the impact of tocopherol and tocotrienol on changes that influence gastric and hormonal parameters important in maintaining gastric mucosal integrity in rats exposed to restrain stress. These include gastric acidity, gastric tissue content of parameters such as malondialdehyde, prostaglandin (PGE(2)), serum levels of gastrin and glucagon-like peptide-1 (GLP-1). Sixty male Sprague-Dawley rats (200-250 g) were randomly divided into three equal sized groups, a control group which received a normal rat diet (RC) and two treatment groups each receiving a vitamin deficient diet with oral supplementation of either tocopherol (TF) or tocotrienol (TT) at 60 mg/kg body weight. Blood samples were taken from half the number of rats (non-stressed group) after a treatment period of 28 days before they were killed. The remaining half was subjected to experimental restraint-stress, at 2 hours daily for 4 consecutive days (stressed groups), on the fourth day, blood samples were taken and the rats killed. The findings showed that the gastric acid concentration and serum gastrin level in stressed rats were significantly (P<0.05) reduced compared to the non-stressed rats in the control and TF groups. However, the gastric acidity and gastrin levels in the TT group were comparable in stressed and non-stressed rats. These findings suggest that tocotrienol is able to preserve the gastric acidity and serum gastrin level which are usually altered in stressed conditions. The PGE(2) content and the plasma GLP-1 level were, however, comparable in all stressed and non-stressed groups indicating that these parameters were not altered in stress and that supplementation with TF or TT had no effect on the gastric PGE2 content or the GLP-1 level. The malondialdehyde, an indicator of lipid peroxidation was higher from gastric tissues in the stressed groups compared to the non-stressed groups. These findings implicated that free radicals may play a role in the development of gastric injury in stress and supplementation with either TF or TT was able to reduce the lipid peroxidation levels compared to the control rats. We conclude that both tocopherol and tocotrienol are comparable in their gastro-protective ability against damage by free radicals generated in stress conditions, but only tocotrienol has the ability to block the stress-induced changes in the gastric acidity and gastrin level.
The term vitamin E denotes a family of tocopherols and tocotrienols, plant lipids that are essential for vertebrate fertility and health. The principal form of vitamin E found in humans, RRR-alpha-tocopherol (TOH), is thought to protect cells by virtue of its ability to quench free radicals, and functions as the main lipid-soluble antioxidant. Regulation of vitamin E homeostasis occurs in the liver, where TOH is selectively retained while other forms of vitamin E are degraded. Through the action of tocopherol transfer protein (TTP), TOH is then secreted from the liver into circulating lipoproteins that deliver the vitamin to target tissues. Presently, very little is known regarding the intracellular transport of vitamin E. We utilized biochemical, pharmacological, and microscopic approaches to study this process in cultured hepatocytes. We observe that tocopherol-HDL complexes are efficiently internalized through scavenger receptor class B type I. Once internalized, tocopherol arrives within approximately 30 min at intracellular vesicular organelles, where it co-localizes with TTP, and with a marker of the lysosomal compartment (LAMP1), before being transported to the plasma membrane in a TTP-dependent manner. We further show that intracellular processing of tocopherol involves a functional interaction between TTP and an ABC-type transporter.
Tocotrienols reverse IKAP and monoamine oxidase deficiencies in familial dysautonomia
Anderson SL, Rubin BY.
Biochem Biophys Res Commun. 2005 Oct 14;336(1):150-6.
Familial dysautonomia (FD), a recessive neurodegenerative disease, is caused by mutations in the IKBKAP gene that result in the production of nonfunctional IKAP protein. Manifestations of FD include autonomic crises characterized by hypertension, tachycardia, diaphoresis, and vomiting. Elevated plasma levels of norepinephrine (NE) and dopamine observed during autonomic crises and an exaggerated hypertensive response to low doses of NE prompted an examination of monoamine oxidase (MAO) levels, key isoenzymes responsible for degrading biogenic and dietary monoamines, in individuals with FD. Fetal tissue homozygous for the common FD-causing mutation and peripheral blood cells of individuals with FD have reduced MAO A mRNA levels. FD-derived cells, stimulated with tocotrienols or EGCG to produce increased levels of functional IKAP, express increased amounts of MAO A mRNA transcript and protein. Administration of tocotrienol to individuals with FD results in increased expression of both functional IKAP and MAO A transcripts in their peripheral blood cells. These findings provide new insight into the pathophysiology of FD and demonstrate the value of therapeutic approaches designed to elevate cellular levels of functional IKAP and MAO A.
The therapeutic impacts of tocotrienols in type 2 diabetic patients with hyperlipidemia
Baliarsingh S, Beg ZH, Ahmad J.
Atherosclerosis. 2005 Oct;182(2):367-74. Epub 2005 Apr 20.
In type 2 diabetics, the progression of atherosclerosis is more rapid than the general population and 80% of these patients will die of an atherosclerotic event. Since in these patients hyperglycemia per se confers increased risk for cardiovascular disease (CVD), the presence of even borderline-high-risk LDL-C signals the need for more aggressive LDL-lowering therapy. Most of the lipid lowering agents, currently in use in the treatment of dyslipidemia in type 2 diabetics, have a host of side effects. In contrast, dietary tocotrienols are Vitamin E and have effective lipid lowering property in addition to their potent antioxidant activity. In this study, we have investigated the therapeutic impacts of tocotrienols on serum and lipoprotein lipid levels in type 2 diabetic patients. Based on known tocotrienol rich fraction (TRF)-mediated decrease on elevated blood glucose and glycated hemoglobin A(1C) (HbA(1C)) in diabetic rats, we have also investigated the effect of TRF on these parameters. A randomized, double blind, placebo-controlled design involving 19 type 2 diabetic subjects with hyperlipidemia was used. After 60 days of TRF treatment, subjects showed an average decline of 23, 30, and 42% in serum total lipids, TC, and LDL-C, respectively. The goal in type 2 diabetics is to reduce LDL-C levels < or = 100mg/dl. In the present investigation tocotrienols mediated a reduction of LDL-C from an average of 179 mg/dl to 104 mg/dl. However, hypoglycemic effect of TRF was not observed in these patients because they were glycemically stable and their glucose and HbA(1) levels were close to normal values. In conclusion, daily intake of dietary TRF by type 2 diabetics will be useful in the prevention and treatment of hyperlipidemia and atherogenesis.
Neuroprotective properties of the natural vitamin E alpha-tocotrienol
Khanna S, Roy S, Slivka A, Craft TK, Chaki S, Rink C, Notestine MA, DeVries AC, Parinandi NL, Sen CK.
Stroke. 2005 Oct;36(10):2258-64. Epub 2005 Sep 15.
BACKGROUND AND PURPOSE: The current work is based on our previous finding that in neuronal cells, nmol/L concentrations of alpha-tocotrienol (TCT), but not alpha-tocopherol (TCP), blocked glutamate-induced death by suppressing early activation of c-Src kinase and 12-lipoxygenase.
METHODS: The single neuron microinjection technique was used to compare the neuroprotective effects of TCT with that of the more widely known TCP. Stroke-dependent brain tissue damage was studied in 12-Lox-deficient mice and spontaneously hypertensive rats orally supplemented with TCT.
RESULTS: Subattomole quantity of TCT, but not TCP, protected neurons from glutamate challenge. Pharmacological as well as genetic approaches revealed that 12-Lox is rapidly tyrosine phosphorylated in the glutamate-challenged neuron and that this phosphorylation is catalyzed by c-Src. 12-Lox-deficient mice were more resistant to stroke-induced brain injury than their wild-type controls. Oral supplementation of TCT to spontaneously hypertensive rats led to increased TCT levels in the brain. TCT-supplemented rats showed more protection against stroke-induced injury compared with matched controls. Such protection was associated with lower c-Src activation and 12-Lox phosphorylation at the stroke site.
CONCLUSIONS: The natural vitamin E, TCT, acts on key molecular checkpoints to protect against glutamate- and stroke-induced neurodegeneration.
The therapeutic impacts of tocotrienols in type 2 diabetic patients with hyperlipidemia
Baliarsingh S, Beg ZH, Ahmad J.
Atherosclerosis. 2005 Oct;182(2):367-74. Epub 2005 Apr 20.
Published
Objectives: In this study, we investigated the therapeutic impacts of tocotrienols on serum and lipoprotein lipid levels in type 2 diabetic patients. Based on known tocotrienol rich fraction (TRF)-mediated decrease on elevated blood glucose and glycated hemoglobin A(1C) (HbA(1C)) in diabetic rats, we have also investigated the effect of TRF on these parameters.
Study design: Randomized, double blind, placebo-controlled design
Subjects: Subjects with type 2 diabetic and hyperlipidemia
Intervention: Tocotrienol-rich fraction versus placebo
Primary outcome: Serum total cholesterol and LDL levels
Methodology: Subjects were first treated to 60 days of TRF treatment after which their serum total lipids, TC and LDL-C were measured.
Results: Subjects showed an average decline of 23, 30, and 42% in serum total lipids, TC, and LDL-C, respectively. The goal in type 2 diabetics is to reduce LDL-C levels < or = 100mg/dl. In the present investigation tocotrienols mediated a reduction of LDL-C from an average of 179 mg/dl to 104 mg/dl. However, hypoglycemic effect of TRF was not observed in these patients because they were glycemically stable and their glucose and HbA(1) levels were close to normal values.
Conclusion: Daily intake of dietary TRF by type 2 diabetics will be useful in the prevention and treatment of hyperlipidemia and atherogenesis.