Nano-Lipids Based on Ginger Oil and Lecithin as a Potential Drug Delivery System.

Lipid nanoparticles based on lecithin are an interesting part of drug delivery systems. However, the stability of lecithin nano-lipids is problematic due to the degradation of lecithin, causing a decrease in pH. In this study, the modification of the conventional nano-lipid-based soybean lecithin was demonstrated. Ginger-oil-derived Zingiber officinale was used along with lecithin, cholesterol and span 80 to fabricate nano-lipids (GL nano-lipids) using a thin-film method. TEM and a confocal microscope were used to elucidate GL nano-lipids' liposome-like morphology. The average size of the resultant nano-lipid was 249.1 nm with monodistribution (PDI = 0.021). The ζ potential of GL nano-lipids was negative, similarly to as-prepared nano-lipid-based lecithin. GL nano-lipid were highly stable over 60 days of storage at room temperature in terms of size and ζ potential. A shift in pH value from alkaline to acid was detected in lecithin nano-lipids, while with the incorporation of ginger oil, the pH value of nano-lipid dispersion was around 7.0. Furthermore, due to the richness of shogaol-6 and other active compounds in ginger oil, the GL nano-lipid was endowed with intrinsic antibacterial activity. In addition, the sulforhodamine B (SRB) assay and live/dead imaging revealed the excellent biocompatibility of GL nano-lipids. Notably, GL nano-lipids were capable of carrying hydrophobic compounds such as curcumin and performed a pH-dependent release profile. A subsequent characterization showed their suitable potential for drug delivery systems.

Changes in ascorbate, glutathione and α-tocopherol concentrations in the brain regions during normal development and moderate hypoglycemia in rats.

Ascorbate, glutathione and α-tocopherol are the major low molecular weight antioxidants in the brain. The simultaneous changes in these compounds during normal development, and under a pro-oxidant condition are poorly understood. Ascorbate, glutathione and α-tocopherol concentrations in the olfactory bulb, cerebral cortex, hippocampus, striatum, hypothalamus, midbrain, cerebellum, pons and medulla oblongata were determined in postnatal day (P) 7, P14 and P60 male rats. A separate group of P14 and P60 rats were subjected to acute hypoglycemia, a pro-oxidant condition, prior to tissue collection. The concentrations of all three antioxidants were 100-600% higher in the brain regions at P7 and P14, relative to P60. The neuron-rich anterior brain regions (cerebral cortex and hippocampus) had higher concentrations of all three antioxidants than the myelin-rich posterior regions (pons and medulla oblongata) at P14 and P60. Hypoglycemia had a differential effect on the antioxidants. Glutathione was decreased at both P14 and P60. However, the decrease was localized at P14 and global at P60. Hypoglycemia had no effect on ascorbate and α-tocopherol at either age. Higher antioxidant concentrations in the developing brain may reflect the risk of oxidant stress during the early postnatal period and explain the relative resistance to oxidant-mediated injury at this age.

Brains of apolipoprotein E deficient mice fed vitamin E deficient diets show alteration in handling alpha tocopherol injected into the cerebral ventricles.

Abnormal function of apolipoprotein E (apoE) has been implicated in the incidence of some neurological disorders including dementia. Our recent experiments have shown that apoE deficiency alters the dynamics of alpha tocopherol (vitamin E) handling by brain. In the current investigation, we examined the uptake and retention of tritium-labeled alpha tocopherol that was injected into the lateral cerebral ventricles of apoE-deficient and wild type mice that were fed vitamin E-deficient diet. Eighteen weeks-old, male mice were fed vitamin E-deficient diets for 28 weeks. Labeled cholesterol was injected with the radioactive tocopherol and the cholesterol counts were used as internal standard. After an equilibration time of 48 h, radioactive alpha tocopherol levels in most brain regions were higher in apoE deficient animals when compared with the wild type. Along with our other data, this suggests that the clearance of vitamin E is slower in apoE-deficient brains. Nearly all of the injected alpha tocopherol was unchanged in the brains of both apoE-deficient and wild type animals (even with the additional dietary stress of vitamin E deficiency) suggesting low turnover rate of tocopherol in brain. The data strongly suggest that apoE is a key protein involved with the transport and/or retention of alpha tocopherol in brain.

Deletion of apolipoprotein E gene modifies the rate of depletion of alpha tocopherol (vitamin E) from mice brains.

Our previous reports show that apolipoprotein E (apoE) influences the dynamics of alpha tocopherol (vitamin E) in brain. In this investigation, the patterns of depletion of alpha tocopherol from tissues of apoE deficient and wild type mice were compared after the animals were fed vitamin E deficient diets. Alpha tocopherol concentrations in specific regions of the brain and peripheral tissues at different times were determined by HPLC with electrochemical detection. ApoE deficiency significantly retarded the rate of depletion of alpha tocopherol from all regions of the brain. In addition, comparison of the rates of depletion of alpha tocopherol in both apoE deficient and wild type animals showed that cerebellum behaved differently from other areas such as cortex, hippocampus and striatum. This reinforces the uniqueness of cerebellum with regard to vitamin E biology. Patterns of depletion of tocopherol from peripheral tissues were different from brain. Serum tocopherol was higher in apoE deficient animals and remained higher than wild type during E deficiency. Depletion of liver tocopherol also tended to be unaffected by apoE deficiency. Our current and previous observations strongly suggest that apoE has an important role in modulating tocopherol concentrations in brain, probably acting in concert with other proteins as well.

Mice lacking alpha-tocopherol transfer protein gene have severe alpha-tocopherol deficiency in multiple regions of the central nervous system.

Ataxia with vitamin E deficiency is caused by mutations in alpha-tocopherol transfer protein (alpha-TTP) gene and it can be experimentally generated in mice by alpha-TTP gene inactivation (alpha-TTP-KO). This study compared alpha-tocopherol (alpha-T) concentrations of five brain regions and of four peripheral organs from 5 months old, male and female, wild-type (WT) and alpha-TTP-KO mice. All brain regions of female WT mice contained significantly higher alpha-T than those from WT males. alpha-T concentration in the cerebellum was significantly lower than that in other brain regions of WT mice. These sex and regional differences in brain alpha-T concentrations do not appear to be determined by alpha-TTP expression which was undetectable in all brain regions. All the brain regions of alpha-TTP-KO mice were severely depleted in alpha-T. The concentration of another endogenous antioxidant, total glutathione, was unaffected by gender but was decreased slightly but significantly in most brain regions of alpha-TTP-KO mice. The results show that both gender and the hepatic alpha-TTP, but not brain alpha-TTP gene expression are important in determining alpha-T concentrations within the brain. Interestingly, functional abnormality (ataxia) develops only very late in alpha-TTP-KO mice in spite of the severe alpha-tocopherol deficiency in the brain starting at an early age.

Apolipoprotein e deficiency leads to altered brain uptake of alpha tocopherol injected into lateral cerebral ventricles.

The incorporation of radioactive alpha tocopherol by various brain regions of wild type and apolipoprotein E (apoE)-deficient mice was investigated. Labeled tocopherol was injected into the lateral cerebral ventricles of 11 weeks old, male mice. Radioactive cholesterol injected simultaneously was used as an internal standard to account for experimental variability. Most areas of the brain of apoE-deficient mice took up less of alpha tocopherol per mg of protein than wild type animals. However, specific activity of alpha tocopherol was higher in cerebellum, pons, hypothalamus, midbrain and cerebral cortex in apoE-deficient brains than the wild type. This could be due to (a) the lower levels of alpha tocopherol in apoE-deficient brain and (b) reductions in the clearance and transport of tocopherol (possibly mediated by apoE). Tocopherol uptake by hippocampus was unusual since it was lower in apoE deficiency whether the data were expressed as specific activity or per mg of protein. Nearly all of the injected alpha tocopherol remained unchanged in the brains of both apoE-deficient and wild type animals suggesting low turnover. Overall, the current data reinforce the hypothesis that apoE is a key protein involved with the transport and/or retention of alpha tocopherol in brain.

Effect of oxidative stress induced by L-dopa on endogenous antioxidants in PC-12 cells.

The mechanism of action of many drugs of abuse involves the dopaminergic pathway. One method of increasing dopamine in brain is by ingestion of L-dopa (3,4-dihydroxy-L-phenylalanine). Interestingly, both dopamine and L-dopa cause oxidative stress which is also a factor in drug-induced damage. Oxidative stress can be reduced by the antioxidant activities of vitamins C (ascorbate) and E (tocopherols). However, the interactions between L-dopa and tocopherols and ascorbate are not well understood. In this article, PC-12 cells (as models of neurons) were cultured for 21 hours with ascorbate (400 microM) and/or alpha tocopherol (25 microM) and in the presence or absence of L-dopa (250 microM). After incubation, cells were harvested and analyzed for the various biochemical components by HPLC. As expected, the addition of L-dopa resulted in an almost threefold increase in cellular dopamine content. Addition of alpha tocopherol resulted in marked increase in cellular tocopherol. Similarly, addition of ascorbate substantially increased its cellular concentration. These increases were strongly attenuated by the presence of L-dopa in the medium. The data indicate that: (a) the uptake systems for ascorbate and tocopherols in these cells are inhibited by L-dopa and/or (b) L-dopa treatment causes an increase in the rate of utilization of the two nutrients. Thus L-dopa modulates the cellular dynamics of ascorbate and tocopherol altering cellular antioxidant protection.

Apolipoprotein E exerts selective and differential control over vitamin E concentrations in different areas of mammalian brain.

Apolipoprotein E (apoE) is known to be a risk factor for the incidence of Alzheimer's disease (AD). In addition, vitamin E has been reported to have a role in the treatment of AD. We examined the potential interrelationship between vitamin E and apoE in brain. As the first step, we determined the concentrations of alpha-tocopherol in selected brain regions of apoE-deficient mice at different ages. The mice were fed normal rodent chow. All regions of the brain in apoE-deficient mice contained less alpha-tocopherol than control samples at 2.5 months of age, the initial time of study. This trend continued for 9.5 months for most regions except the spinal cord and cerebellum. Tocopherol levels in these latter regions of apoE-deficient animals increased to control levels during the study. Serum alpha-tocopherol and cholesterol levels were high in the apoE-deficient animals; however, the CNS cholesterol levels were the same in apoE-deficient and control mice. This suggests that 1) the decline in brain alpha-tocopherol in apoE deficiency is not due to overall alterations in lipid metabolism; and 2) the processing of alpha-tocopherol in brain follows a separate pathway than that of cholesterol. Subcellular concentrations of alpha-tocopherol were unaltered by apoE deficiency indicating that intracellular handling of tocopherol is not affected by apoE. ApoE may be an important protein controlling vitamin E levels in specific brain regions. Further understanding of the interactions between apoE and vitamin E could be important in the appropriate use of vitamin E in AD.

Moderate hyperoxia (40%) increases antioxidant levels in mouse tissue.

BACKGROUND: Oxygen is routinely administered to patients to improve clinical outcome. Since studies have shown that administering 100% oxygen can cause unwanted side effects, intermediate concentrations of 40% oxygen are used in clinical practice. In this study, we examined whether the breathing of 40% oxygen causes beneficial effects upon tissue levels of antioxidants such as vitamin E, vitamin C, and glutathione. METHODS: Four-month-old mice were separated into two groups: control (n = 11) and experimental (n = 11). The treatment group was administered 40% oxygen for 10 days. Brain, heart, lung, liver, testes, and skeletal muscle were harvested and tissue antioxidant levels were determined by HPLC. RESULTS: Vitamin E concentrations were higher in brain, heart, lung, liver, and testes of the treatment group (P < 0.05). Glutathione concentrations were higher in the lung tissue only (P < 0.05). No differences were found in vitamin C levels. CONCLUSIONS: The data suggest that mice respond to oxidative stress by increasing tissue vitamin E incorporation and cellular synthesis of glutathione in the lung when exposed to moderate levels (40%) of hyperoxia.

Oxidative stress and inhibition of oxidative phosphorylation induced by peroxynitrite and nitrite in rat brain subcellular fractions.

Nitrite and nitrate, two endogenous oxides of nitrogen, are toxic in vivo. Furthermore, the reaction of superoxide (produced by all aerobic cells) with nitric oxide (NO) generates peroxynitrite, a potent oxidizing agent, that can cause biological oxidative stress. Using subcellular fractions from rat brain hemispheres we studied oxidative stress induced by these nitrogen compounds with special emphasis on nitrite. The consumption of Vitamin C (ascorbate) and Vitamin E (alpha tocopherol), two of the important nutritional antioxidants, was followed in synaptosomes (nerve-ending particles) and mitochondria along with changes in parameters of mitochondrial oxidative phosphorylation. Nitrite, but not nitrate, oxidized ascorbate without oxidizing alpha tocopherol in both synaptosomes and mitochondria whereas peroxynitrite oxidized both ascorbate and alpha tocopherol. Functionally, both nitrite and peroxynitrite inhibited mitochondrial oxidative phosphorylation. Nitrite was less potent than peroxynitrite when the effects of equal concentrations of the two were compared. However, since nitrite is much more stable than peroxynitrite the impact of nitrite as an oxidant in vivo could be as much or even more significant than peroxynitrite. Nitrate would not have similar action unless it is reduced to nitrite. It is possible that nitrite may impair oxidative phosphorylation through modulating levels of nitric oxide, changing the activity of heme proteins or a mild uncoupling of mitochondria.

Alpha and gamma tocopherols in cerebrospinal fluid and serum from older, male, human subjects.

OBJECTIVE: The major forms of vitamin E in human physiological fluids are alpha and gamma tocopherols which exhibit different biological activities under a variety of assay conditions. The goal of this study was to obtain indirect information about the transport of tocopherols across the blood/spinal fluid barrier by comparing the concentrations of alpha and gamma tocopherols in serum and cerebrospinal fluid (CSF). METHODS: CSF and serum samples were obtained simultaneously from 28 human, male subjects excluding those with known pathology during the performance of spinal anesthesia procedures. The samples were centrifuged and frozen, and analyzed for tocopherols by HPLC with electrochemical detection. RESULTS: The concentrations of alpha and gamma tocopherols in CSF correlated significantly with their respective concentrations in serum. This would be expected since these nutrients have to be supplied by diet to serum followed by transport to the brain. The ratios of alpha to gamma tocopherols in the CSF and serum were highly correlated. High concentrations of alpha in serum tended to suppress gamma in both serum and CSF. CONCLUSIONS: These data suggest that the processes involved in the entry of tocopherol from blood to the CSF do not discriminate between the alpha and gamma tocopherols. In contrast, alpha tocopherol is highly preferred during the packaging of plasma lipoproteins by the liver. Our data also suggest that alpha and gamma tocopherols will be available to the human brain via transport from blood.

Iron uncouples oxidative phosphorylation in brain mitochondria isolated from vitamin E-deficient rats.

Few, if any, studies have examined the effect of vitamin E deficiency on brain mitochondrial oxidative phosphorylation. The latter was studied using brain mitochondria isolated from control and vitamin E-deficient rats (13 months of deficiency) after exposure to iron, an inducer of oxidative stress. Mitochondria were treated with iron (2 to 50 microM) added as ferrous ammonium sulfate. Rates of state 3 and state 4 respiration, respiratory control ratios, and ADP/O ratios were not affected by vitamin E deficiency alone. However, iron uncoupled oxidative phosphorylation in vitamin E-deficient mitochondria, but not in controls. In vitamin E-deficient mitochondria, iron decreased ADP/O ratios and markedly stimulated state 4 respiration; iron had only a modest effect on these parameters in control mitochondria. Thus, vitamin E may have an important role in sustaining oxidative phosphorylation. Low concentrations of iron (2 to 5 microM) oxidized mitochondrial tocopherol that exists in two pools. The release of iron in brain may impair oxidative phosphorylation, which would be exacerbated by vitamin E deficiency. The results are important for understanding the pathogenesis of human brain disorders known to be associated with abnormalities in mitochondrial function as well as iron homeostasis (e.g., Parkinson's disease).

Oxidation of vitamin E and vitamin C and inhibition of brain mitochondrial oxidative phosphorylation by peroxynitrite.

The effects of peroxynitrite (PN; product of the reaction between nitric oxide and superoxide) on mitochondrial respiration as well as oxidation of alpha-tocopherol and ascorbic acid were studied. Mitochondria were isolated from brain hemispheres of 4-month-old male Fisher rats by standard centrifugation procedures utilizing Ficoll gradients. Treatment of brain mitochondria with PN caused a concentration-dependent impairment of oxidative phosphorylation and depletion of the endogenous antioxidants alpha-tocopherol and ascorbic acid. PN-induced mitochondrial dysfunction was characterized by 1) decreases in state 3 respiration and oxidative phosphorylation, 2) loss of respiratory control [ratio of ADP-stimulated (state 3) to basal (state 4) respiration], and 3) uncoupling of oxidative phosphorylation. PN did not function as a pure uncoupler, insofar as the increase in state 4 respiration was accompanied by a larger decrease in state 3 respiration. This contrasts with the uncoupling action of the protonophore carbonyl cyanide m-chlorophenylhydrozone, which increases both state 3 and state 4 respiration. PN-induced reduction in respiratory control and oxidative phosphorylation closely paralleled the oxidation of membrane tocopherol and were preceded by loss of ascorbate. alpha-Tocopherol (the most potent biological lipid antioxidant) may have a unique role in protecting mitochondrial membranes from oxidative stress. The two antioxidant nutrients alpha-tocopherol and ascorbate (which interact with each other and glutathione) may be intimately involved in protecting mitochondria in situations in which excessive release of superoxide and nitric oxide occurs under normal and/or pathological conditions.

Effect of vitamin E deficiency on the growth and secretory function of the rat prostatic complex.

Increased intake of vitamin E has been suggested to be protective against prostate cancer in men, but the effects of vitamin E on prostate growth and function remain poorly defined. The purpose of this study was to determine the effects of vitamin E deficiency on pubertal growth and maturation of the prostate in the rat. Animals were placed on a vitamin E deficient diet at 28 days of age and were followed for 15 and 26 weeks. Vitamin E deficient rats had a circulating vitamin E level of less than 1% of control animals and experienced a decrease in body and testis weight. The deficiency did not alter the weights of the ventral and dorsal lobes of the prostate. However, there was an increase in weight, DNA, and protein contents of the lateral lobe in control and vitamin E deficient rats from 15 to 26 weeks of treatment, but these increases were significantly lower in vitamin E deficient 26-week treated rats. The volume of secretion per milligram tissue was greater in the ventral than lateral or dorsal lobes. The volume of secretion and activity of the secretory 26 kDa protease in the ventral prostate was lower in vitamin E deficient rats at 15 weeks, but not at 26 weeks of treatment. In contrast, the relative protein content of lateral lobe secretion increased in both control and vitamin E deficient rats from 15 to 26 weeks of treatment. The lateral, but not ventral or dorsal, lobes of both control and vitamin E deficient rats were affected by chronic prostatitis as evidenced by infiltration of inflammatory cells. The lateral lobes also showed markedly elevated activities of the matrix metalloproteinases gelatinase A (MMP-2) and gelatinase B (MMP-9). These data indicate that vitamin E deficiency does not alter the growth of the prostatic lobes, nor the onset and extent of lateral lobe specific prostatitis, but it may delay some differentiated functions such as secretion of specific proteins in the ventral lobe. Thus, the effects of vitamin E in the prostate of the rat appear to be selective.