Supplementation with Eurycoma longifolia Extract Modulates Diurnal Body Temperature Fluctuation and Sleep Rhythm in Mice.

Eurycoma longifolia (Tongkat Ali; TA) is a traditional medicinal herb, commonly known as Malaysian ginseng. The root tea has been traditionally applied to treat fevers, aches, sexual dysfunction and other ailments. We evaluated the effects of TA extract supplementation on diurnal core body temperature (BT) and sleep architecture in model mice. Dietary supplementation with TA extract for 4 wk resulted in significantly and moderately reduced BT during the rest and active phases, respectively. A high dose delayed the onset of BT elevation at the start of the active phase, indicating that the effect was dose-dependent. Electroencephalography findings revealed that dietary supplementation with TA extract changed sleep rhythms and delta power during the inactive phase of NREM sleep, indicating improved sleep quality. Our findings suggested that dietary TA extract could be a promising natural aid that alleviates sleep problems via thermoregulation.

Wheat alkylresorcinols suppress high-fat, high-sucrose diet-induced obesity and glucose intolerance by increasing insulin sensitivity and cholesterol excretion in male mice.

BACKGROUND: Epidemiologic studies have shown that the consumption of whole grains can reduce the risk of type 2 diabetes mellitus, cardiovascular disease, and all-cause mortality. However, the underlying mechanisms remain a matter of debate. OBJECTIVE: We aimed to determine the effects of wheat bran-derived alkylresorcinols on diet-induced metabolic disorders in mice. METHODS: We fed C57BL/6J mice a normal refined diet or a high-fat, high-sucrose diet [29.1% fat, 20.7% protein, 34.0% carbohydrates containing 20.0% sucrose (w/w)] alone (FS) or containing 0.4% (wt:wt) alkylresorcinols (FS-AR) for 10 wk. RESULTS: The alkylresorcinols suppressed FS-induced increases in body weight by 31.0% as well as FS-induced hepatic triglyceride accumulation (means ± SEMs: 29.6 ± 3.18 and 19.8 ± 2.42 mg/g tissue in the FS and FS-AR groups, respectively), without affecting energy intake. We measured circadian changes in blood metabolic hormones and found that FS-induced hyperinsulinemia (5.1 and 2.1 µg/L at night in the FS and FS-AR groups, respectively) and hyperleptinemia (21.6 and 10.8 µg/L at night in the FS and FS-AR groups, respectively) were suppressed by alkylresorcinols. Glucose and insulin tolerance tests showed that alkylresorcinols significantly reduced fasting blood glucose concentrations (190 ± 3.62 and 160 ± 8.98 mg/dL in the FS and FS-AR groups, respectively) and suppressed glucose intolerance as well as insulin resistance induced by the FS diet. Furthermore, alkylresorcinols significantly increased insulin-stimulated hepatic serine/threonine protein kinase B phosphorylation compared to the FS diet (+81.3% and +57.4% for Ser473 and Thr308, respectively). On the other hand, pyruvate and starch tolerance tests suggested that alkylresorcinols did not affect gluconeogenesis and carbohydrate digestion, respectively. Alkylresorcinols significantly increased fecal cholesterol excretion by 39.6% and reduced blood cholesterol concentrations by 30.4%, while upregulating the expression of hepatic cholesterol synthetic genes such as sterol regulatory element binding protein 2 (Srebf2) and 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (Hmgcs1). CONCLUSIONS: These findings suggest that wheat alkylresorcinols increase glucose tolerance and insulin sensitivity by suppressing hepatic lipid accumulation and intestinal cholesterol absorption, which subsequently suppresses diet-induced obesity in mice.

Harmine lengthens circadian period of the mammalian molecular clock in the suprachiasmatic nucleus.

The circadian clock is a cell-autonomous endogenous system that generates circadian rhythms in the behavior and physiology of most organisms. We previously reported that the harmala alkaloid, harmine, lengthens the circadian period of Bmal1 transcription in NIH 3T3 fibroblasts. Clock protein dynamics were examined using real-time reporter assays of PER2::LUC to determine the effects of harmine on the central clock in the suprachiasmatic nucleus (SCN). Harmine significantly lengthened the period of PER2::LUC expression in embryonic fibroblasts, in neuronal cells differentiated from neuronal progenitor cells and in SCN slices obtained from PER2::LUC mice. Although harmine did not induce the transient mRNA expression of clock genes such as Per1, Per2 and Bmal1 in embryonic fibroblasts, it significantly extended the half-life of PER2::LUC protein in neuronal cells and SCN slices. Harmine might lengthen the circadian period of the molecular clock by increasing PER2 protein stability in the SCN.

Dietary heat-killed Lactobacillus brevis SBC8803 promotes voluntary wheel-running and affects sleep rhythms in mice.

AIMS: We previously reported that heat-killed Lactobacillus brevis SBC8803 enhances appetite via changes in autonomic neurotransmission. Here we assessed whether a diet supplemented with heat-killed SBC8803 affects circadian locomotor rhythmicity and sleep architecture. MAIN METHODS AND KEY FINDINGS: Daily total activity gradually increased in mice over 4 weeks and supplementation with heat-killed SBC8803 significantly intensified the increase, which reached saturation at 25 days. Electroencephalography revealed that SBC8803 supplementation significantly reduced the total amount of time spent in non-rapid eye movement (NREM) sleep and increased the amount of time spent being awake during the latter half of the nighttime, but tended to increase the total amount of time spent in NREM sleep during the daytime. Dietary supplementation with SBC8803 can extend the duration of activity during the nighttime and of sleep during the daytime. Daily voluntary wheel-running and sleep rhythmicity become intensified when heat-killed SBC8803 is added to the diet. SIGNIFICANCE: Dietary heat-killed SBC8803 can modulate circadian locomotion and sleep rhythms, which might benefit individuals with circadian rhythms that have been disrupted by stress or ageing.

Disruption of behavioral circadian rhythms induced by psychophysiological stress affects plasma free amino acid profiles without affecting peripheral clock gene expression in mice.

Disordered circadian rhythms are associated with various psychiatric conditions and metabolic diseases. We recently established a mouse model of a psychophysiological stress-induced chronic sleep disorder (CSD) characterized by reduced amplitude of circadian wheel-running activity and sleep-wake cycles, sleep fragmentation and hyperphagia. Here, we evaluate day-night fluctuations in plasma concentrations of free amino acids (FAA), appetite hormones and prolactin as well as the hepatic expression of circadian clock-related genes in mice with CSD (CSD mice). Nocturnal increases in wheel-running activity and circadian rhythms of plasma prolactin concentrations were significantly disrupted in CSD mice. Hyperphagia with a decreased leptin/ghrelin ratio was found in CSD mice. Day-night fluctuations in plasma FAA contents were severely disrupted without affecting total FAA levels in CSD mice. Nocturnal increases in branched-chain amino acids such as Ile, Leu, and Val were further augmented in CSD mice, while daytime increases in Gly, Ala, Ser, Thr, Lys, Arg, His, Tyr, Met, Cys, Glu, and Asn were significantly attenuated. Importantly, the circadian expression of hepatic clock genes was completely unaffected in CSD mice. These findings suggest that circadian clock gene expression does not always reflect disordered behavior and sleep rhythms and that plasma FFA profiles could serve as a potential biomarker of circadian rhythm disorders.

Effects of intraduodenal injection of Lactobacillus brevis SBC8803 on autonomic neurotransmission and appetite in rodents.

Lactobacilli provide several health benefits to mammals, including humans. We previously observed that in rats, intraduodenal injection of Lactobacillus johnsonii La1 elevated efferent gastric vagal nerve activity (efferent-GVNA), while Lactobacillus paracasei ST11 suppressed efferent-GVNA, and thereby increased or decreased food intake. To determine the function of Lactobacillus brevis (SBC8803), its effect on food intake was examined by providing food containing heat-killed SBC8803 to mice. We observed that administration of SBC8803 elevated food intake. Because the afferent intestinal vagal nerve (IVN) is hypothesized to be involved in efferent-GVNA changes, we examined the effect of intraduodenal administration of heat-killed SBC8803 on efferent-GVNA and afferent-IVN activity (IVNA) in rats. In this study, we found that intraduodenal administration of heat-killed SBC8803 increased both efferent-GVNA and afferent-IVNA in rats. Moreover, IV administration of the serotonin 3 receptor antagonist granisetron eliminated the effects of SBC8803 on efferent-GVNA and afferent-IVNA. These findings suggest that heat-killed SBC8803 enhances appetite by elevating digestion and absorption abilities via changes in autonomic neurotransmission that might be mediated by the serotonin 3 receptor.

Continuous exposure to a novel stressor based on water aversion induces abnormal circadian locomotor rhythms and sleep-wake cycles in mice.

Psychological stressors prominently affect diurnal rhythms, including locomotor activity, sleep, blood pressure, and body temperature, in humans. Here, we found that a novel continuous stress imposed by the perpetual avoidance of water on a wheel (PAWW) affected several physiological diurnal rhythms in mice. One week of PAWW stress decayed robust circadian locomotor rhythmicity, while locomotor activity was evident even during the light period when the mice are normally asleep. Daytime activity was significantly upregulated, whereas nighttime activity was downregulated, resulting in a low amplitude of activity. Total daily activity gradually decreased with increasing exposure to PAWW stress. The mice could be exposed to PAWW stress for over 3 weeks without adaptation. Furthermore, continuous PAWW stress enhanced food intake, but decreased body weight and plasma leptin levels, indicating that sleep loss and PAWW stress altered the energy balance in these mice. The diurnal rhythm of corticosterone levels was not severely affected. The body temperature rhythm was diurnal in the stressed mice, but significantly dysregulated during the dark period. Plasma catecholamines were elevated in the stressed mice. Continuous PAWW stress reduced the duration of daytime sleep, especially during the first half of the light period, and increased nighttime sleepiness. Continuous PAWW stress also simultaneously obscured sleep/wake and locomotor activity rhythms compared with control mice. These sleep architecture phenotypes under stress are similar to those of patients with insomnia. The stressed mice could be entrained to the light/dark cycle, and when they were transferred to constant darkness, they exhibited a free-running circadian rhythm with a timing of activity onset predicted by the phase of their entrained rhythms. Circadian gene expression in the liver and muscle was unaltered, indicating that the peripheral clocks in these tissues remained intact.

Disrupted daily light-dark cycle induces the expression of hepatic gluconeogenic regulatory genes and hyperglycemia with glucose intolerance in mice.

We elucidated associations between metabolic disorders and the environmental light-dark (LD) cycle that entrains the circadian clock located in the suprachiasmatic nucleus of mammals. Mice were fed with a high-fat/high-sucrose diet for eight weeks under a normal 12h light-12h dark cycle (LD 12:12) or an ultradian 3h light-3h dark cycle (LD 3:3) that might perturb the central clock. The circadian behavioral rhythms were gradually disturbed under LD 3:3. Hyperglycemia with glucose intolerance and increases in diabetic markers, glycated albumin and hemoglobin A1c, were significantly induced without affecting body weight gain and food consumption in LD 3:3. Expression levels of hepatic gluconeogenic regulatory genes such as Pck1, G6pc, Hnf4a, and Foxo1/3/4 genes were increased under LD 3:3. Hypercholesterolemia with hepatic cholesterol accumulation was also induced in LD 3:3. Ultradian LD 3:3 cycles did not affect the adipose inflammation that is considered a major player in obesity-associated metabolic disorders. Our findings provide a link between metabolic disorders and environmental photoperiodic cycles in genetically normal animals.

Low-carbohydrate, high-protein diet affects rhythmic expression of gluconeogenic regulatory and circadian clock genes in mouse peripheral tissues.

Recent studies have demonstrated that metabolic changes in mammals induce feedback regulation of the circadian clock. The present study evaluates the effects of a low-carbohydrate high-protein diet (HPD) on circadian behavior and peripheral circadian clocks in mice. Circadian rhythms of locomotor activity and core body temperature remained normal in mice fed with the HPD diet (HPD mice), suggesting that it did not affect the central clock in the hypothalamus. Two weeks of HPD feeding induced mild hypoglycemia without affecting body weight, although these mice consumed more calories than mice fed with a normal diet (ND mice). Plasma insulin levels were increased during the inactive phase in HPD mice, but increased twice, beginning and end of the active phase, in ND mice. Expression levels of the key gluconeogenic regulatory genes PEPCK and G6Pase were significantly induced in the liver and kidneys of HPD mice. The HPD appeared to induce peroxisome proliferator-activated receptor α (PPARα) activation, since mRNA expression levels of PPARα and its typical target genes, such as PDK4 and Cyp4A10, were significantly increased in the liver and kidneys. Circadian mRNA expression of clock genes, such as BMAL1, Cry1, NPAS2, and Rev-erbα, but not Per2, was significantly phase-advanced, and mean expression levels of BMAL1 and Cry1 mRNAs were significantly elevated, in the liver and kidneys of HPD mice. These findings suggest that a HPD not only affects glucose homeostasis, but that it also advances the molecular circadian clock in peripheral tissues.

PPARγ activation induces acute PAI-1 gene expression in the liver but not in adipose tissues of diabetic model mice.

Insulin resistance in patients with type II diabetes has recently been treated with thiazolidinediones, a class of peroxisome proliferator-activated receptor γ (PPARγ) agonists. However, these compounds are possibly associated with a significant increase in the risk of cardiovascular events. We examined the effect of the PPARγ agonist rosiglitazone on the expression of plasminogen activator inhibitor-1 (PAI-1) that is the primary inhibitor of fibrinolysis in the liver of diabetic mice and cultured mouse and human hepatocytes. Concentrations of plasma PAI-1 and levels of its mRNA expression in the liver were significantly elevated in accordance with hepatic PPARγ1 and PPARγ2 mRNA accumulation in genetically diabetic db/db mice. An intraperitoneal injection of rosiglitazone significantly increased plasma PAI-1 concentrations in parallel with hepatic, but not with adipose mRNA levels in db/db mice, and did not affect these parameters in wild-type mice. Rosiglitazone as well as the PPARα agonist bezafibrate significantly induced PAI-1 mRNA expression in cultured mouse hepatocytes. Furthermore, both rosiglitazone and pioglitazone significantly induced, whereas bezafibrate did not affect PAI-1 mRNA expression in the human liver carcinoma cell line HepG2. The transient induction of PAI-1 gene expression mediated by PPARγ in the fatty liver might be involved in the increased risk of cardiovascular events associated with thiazolidinediones in diabetic patients through decreasing fibrinolytic activity.

The role of alpha-tocopherol in motor hypofunction with aging in alpha-tocopherol transfer protein knockout mice as assessed by oxidative stress biomarkers.

It has been hypothesized that oxidative stress plays a key role in aging. In order to elucidate the role of the antioxidant network - including alpha-tocopherol (alphaT) and alphaT transfer protein - in aging in vivo, alpha-tocopherol transfer protein knockout (alphaTTP(-/-)) mice were fed a vitamin-E-depleted diet, and wild-type (WT) mice were fed a diet containing 0.002 wt.% alphaT from the age of 3 months to 1 1/2 years. The lipid oxidation markers total hydroxyoctadecadienoic acid (tHODE) and 8-iso-prostaglandin F(2)alpha, and antioxidant levels in the blood, liver and brain were measured at 3, 6, 12 and 18 months. tHODE levels in the plasma of alphaTTP(-/-) mice were elevated at 6 months compared to 3 months, and were significantly higher those in WT mice, although they decreased thereafter. On the other hand, tHODE levels in the liver and brain were constantly higher in alphaTTP(-/-) mice than in WT mice. Motor activities decreased with aging in both mouse types; however, those in the alphaTTP(-/-) mice were lower than those in the WT mice. It is intriguing to note that motor activities were significantly correlated with the stereoisomer ratio (Z,E/E,E) of HODE, which is a measure of antioxidant capacity in vivo, in the plasma, in the liver and even in the brain, but not with other factors such as antioxidant levels. In summary, using the biomarker tHODE and its stereoisomer ratio, we demonstrated that alphaT depletion was associated with a decrease in motor function, and that this may be primarily attributable to a decrease in the total antioxidant capacity in vivo.

Antioxidant effects of 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran and alpha-tocopherol in hyperlipidemic mice as evaluated by hydroxyoctadecadienoic acid and 7-hydroxycholesterol.

It has been hypothesized that oxidative modification of low density lipoprotein plays a key role in the pathogenesis of atherosclerosis. In order to elucidate the role of lipid oxidation and its inhibition in vivo, apolipoprotein E and alpha-tocopherol (alphaT) transfer protein double knockout (ApoE(-/-)alpha-TTP(-/-)) and apolipoprotein E knockout (ApoE(-/-)) mice fed with a vitamin E-depleted diet and a diet containing 0.002 wt.% alphaT, respectively, were used with or without the treatment of a synthetic antioxidant 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran (BO-653, 0.2 wt.%). The lipid oxidation markers of total hydroxylinoleic acid (tHODE), 8-iso-prostaglandin F(2alpha), and 7-hydroxycholesterol (t7-OHCh) in the blood, liver, and brain were inclusively measured with or without an excessive cholesterol-feeding (Ch-diet). The tHODE levels were elevated by Ch-diet in the plasma and brain of ApoE(-/-)alpha-TTP(-/-) mice and in the liver of ApoE(-/-) mice without BO-653. The levels of t7-OHCh in the liver were also increased due to the Ch-diet, and the ratio of t7-OHCh to the parent compound cholesterol was reduced to the control levels by BO-653. In summary, it was demonstrated by biomarkers, tHODE and t7-OHCh, that the added BO-653 in their diets exerted antioxidative effects in vivo under the condition of reduced vitamin E.

Evaluation of lipophilic antioxidant efficacy in vivo by the biomarkers hydroxyoctadecadienoic acid and isoprostane.

The evaluation of antioxidant activity in vivo is difficult. In this study, the effects of dietary natural and synthetic antioxidants on the lipid peroxidation in mice were assessed using a biomarker, total hydroxyoctadecadienoic acid (tHODE). Biological samples such as plasma, erythrocytes, and tissues were first reduced and then saponified to convert various oxidation products of linoleates to tHODE. Subsequently, the absolute concentration of tHODE and its stereoisomer ratio, [9- and 13-(Z,E)-HODE)/[9- and 13-(E,E)-HODE], which is a measure of the hydrogen donor capacity of antioxidants, were determined by gas chromatography-mass spectrometry (GC-MS) analyses. These were then compared with total 8-iso-prostaglandin F(2alpha) (t8-iso-PGF(2alpha)) which was also assessed after reduction and saponification. Remarkable increases in tHODE and t8-iso-PGF(2alpha) levels were observed in the plasma, erythrocytes, liver, and brain of mice that were fed an alpha-tocopherol (alphaT)-stripped (E-free) diet for 1 month when compared with those of mice that were fed a standard diet (alphaT = 0.002 wt%). When mice were fed for 1 month on an E-free diet supplemented with a lipophilic antioxidant (0.04 wt%), namely, alphaT, alpha-tocotrienol (alphaT3), gamma-tocopherol (gammaT), or 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran (BO-653), a potent synthetic antioxidant, the increases of tHODE and t8-iso-PGF(2alpha) in the plasma, erythrocytes, liver, and brain were suppressed to the levels lower than those of mice fed a standard diet. The (Z,E/E,E) HODE ratio was decreased in the plasma and erythrocytes of mice fed the E-free diet when compared with that in mice fed the standard diet. This stereo-isomeric ratio was significantly recovered by the addition of alphaT and BO-653. These results show that the tHODE level and the (Z,E/E,E) HODE ratio are useful biomarkers for the assessment of antioxidant capacity in vivo and that the antioxidant capacity decreased in the order: BO-653 > alphaT3 >or= alphaT, gammaT, as assessed by tHODE levels from blood, liver, and brain.

Advantages and limitation of BODIPY as a probe for the evaluation of lipid peroxidation and its inhibition by antioxidants in plasma.

Oxidative stress and the role of antioxidants are currently one of the most important subjects in the field of life science. In the present study, we assessed the oxidation of plasma lipids induced by free radicals and its inhibition by antioxidants with a fluorescence probe BODIPY. Vitamin E and C-depleted plasma was used to evaluate the inherent action of several antioxidants. BODIPY reacted with free radicals in plasma to emit fluorescence (ex. 510 nm, em. 520 nm), which was suppressed by the antioxidants in a concentration-dependent manner. However, the suppression of fluorescence emission by antioxidants did not always correlate quantitatively with the suppression of lipid peroxidation. For example, alpha-tocopherol suppressed BODIPY fluorescence but enhanced the peroxidation of plasma lipids in the absence of ascorbic acid. 2,2,5,7,8-Pentamethyl-6-chromanol, a vitamin E analogue without a phytyl side chain, almost completely suppressed both fluorescence emission and lipid peroxidation in the plasma. These results show that BODIPY can be used as a convenient probe for radical scavenging, but that care should be taken for the evaluation of antioxidant capacity.

gamma-Tocopherol attenuates MPTP-induced dopamine loss more efficiently than alpha-tocopherol in mouse brain.

Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mouse has been widely used as a rodent model of Parkinson's disease. In this study, alpha-tocopherol (alphaT) transfer protein knockout (heteromutant type, alpha-TTP((+/-))) mice were used to evaluate the protective effects of alphaT and gamma-tocopherol (gammaT) against MPTP-induced neurotoxicity. The intraperitoneal administration of MPTP to mice induced a decrease in the striatal levels of dopamine (DA) 3 days after the administration in both alpha-TTP((+/-)) and wild-type mice; these mice were fed an alphaT-deficient diet for 3 weeks before the MPTP administration. The DA levels in the alpha-TTP((+/-)) mice, which had been fed a gammaT-fortified diet (0.10 wt.%) for 3 weeks and were administered with MPTP, were recovered to those of the control, whereas there was no significant protective effect of alphaT despite the considerably higher striatal concentration of alphaT than gammaT. The immunohistochemical study also revealed that gammaT exerted a protective effect against neurodegenerative toxicity of MPTP. Collectively, this is the first report showing that the protective effect of gammaT is stronger than that of alphaT against the MPTP-induced damage of dopaminergic neurons in the mouse.

Lipid peroxidation in mice fed a choline-deficient diet as evaluated by total hydroxyoctadecadienoic acid.

OBJECTIVE: The relevance of oxidative stress in mice fed a choline-deficient diet (CDD) was investigated in relation to the oxidative stress marker, hydroxyoctadecadienoic acid (HODE) in comparison with F2-isoprostanes. Further, the protective effects of antioxidants against oxidative damage were assessed by using HODE. METHODS: We recently proposed total HODE as a biomarker for oxidative stress in vivo. Biological samples such as plasma, urine, and tissues were first reduced and then saponified to convert various oxidation products of linoleates to HODE. In the present study, this method was applied to measure oxidative damage in mice induced by CDD for 1 mo. RESULTS: CDD, when compared with choline-controlled diet (CCD), increased liver weight and fatty acid accumulation but the increase in body weight was less significant. Remarkable increases in HODE and 8-iso-prostaglandin F(2alpha) in liver and plasma were observed when mice were fed with the CDD for 1 mo compared with the CCD. The HODE level was about two to three orders higher than the F2-isoprostane level. This increase was decreased to the level of the CCD when alpha-tocopherol or 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran, a potent synthetic antioxidant, was mixed with the CDD. The stereoisomer ratio of HODE (9-and-13 (Z,E)-HODE/9-and-13 (E,E)-HODE) was decreased by CDD compared with CCD, which was spared by the addition of alpha-tocopherol and 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran. However, the increase in plasma glutamic-pyruvic transaminase and fatty acids in liver induced by the CDD was not recovered by any antioxidant. CONCLUSIONS: This study clearly demonstrated that oxidative stress was involved in fatty liver formation induced by the CDD and that HODE was a good biomarker for an oxidative stress in vivo.

Lipid peroxidation induced by carbon tetrachloride and its inhibition by antioxidant as evaluated by an oxidative stress marker, HODE.

We have recently proposed total hydroxyoctadecadienoic acid (HODE) as a biomarker for oxidative stress in vivo. The biological samples such as plasma, urine, and tissues were first reduced and then saponified to convert the oxidation products of linoleate to HODE. In the present study, this method was applied to measure the oxidative damage induced by the administration of carbon tetrachloride to mice and also to evaluate the capacity of antioxidant to inhibit the above damage. alpha-Tocopherol transfer protein knock out (alpha-TTP-/-) mice were used to evaluate antioxidant effect in the absence of alpha-tocopherol. The intraperitoneal administration of carbon tetrachloride to mice induced the increase in HODE in liver and plasma, which was followed by an increase in plasma glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT). F2-isoprostanes, another prevailing biomarker, were also increased similarly, but their concentration was approximately two to three orders of magnitude smaller than that of HODE. The lipophilic antioxidants such as gamma-tocopherol, gamma-tocotrienol and 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran (BO-653) were effective in suppressing the formation of HODE.

Distribution of tocopherols and tocotrienols to rat ocular tissues after topical ophthalmic administration.

With increasing evidence suggesting the involvement of oxidative stress in various disorders and diseases, the role of antioxidants in vivo has received much attention. Chemically, tocopherols and tocotrienols are closely related; however, it has been observed that they have widely varying degrees of biological effectiveness. The present study has been carried out in an attempt to deepen our understanding of whether there is a significant difference in distribution between tocopherol and tocotrienol homologs to rat eye tissues. Rats were administered 5 microL of pure tocopherol or tocotrienol to each eye once a day for 4 d. Various tissues of the eyes were separated and analyzed for tocopherol and tocotrienol concentrations. The concentration of alpha-to-cotrienol increased markedly in every tissue to which it was administered; however, no significant increase was observed in the case of alpha-tocopherol. The intraocular penetration of gamma-tocopherol and gamma-tocotrienol did not differ significantly. Additionally, a significant increase in total vitamin E concentration was observed in ocular tissues, including crystalline lens, neural retina, and eye cup, with topical administration using a relatively small amount (5 microL) of vitamin E, whereas no significant increase was observed when the same amount of vitamin E was administered orally. Topical administration of tocotrienols is thus an effective way to increase ocular tissue vitamin E concentration.

Inhibition of plasma lipid peroxidation by anti-atherogenic antioxidant BO-653, 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran.

BO-653, 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran, is a synthetic antioxidant which is now being developed as an anti-atherogenic drug. The antioxidant action of BO-653 against lipid peroxidation in rat plasma was studied and compared with its analogue BO-653M, 2,3-dihydro-5-hydroxy-4,6-di-methyl-2,2-dipentylbenzofuran, and vitamin E. BO-653 was readily incorporated into plasma by oral administration and it inhibited plasma lipid peroxidation more efficiently than vitamin E independent of the presence or absence of vitamin C. On the other hand, its analogue BO-653M having two methyl substituents in place of tert-butyl groups of BO-653 did not inhibit the lipid peroxidation in plasma as efficiently as BO-653, demonstrating clearly that the tert-butyl groups at the ortho-position play a key role in determining the antioxidant efficacy.

Application of water-soluble radical initiator, 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride, to a study of oxidative stress.

It is essential to generate free radicals at a controled and constant rate for specific duration and at specific site to study the dynamics of oxidation and also antioxidation. Both hydrophilic and lipophilic azo compounds have been used for such purpose. In the present work, the action of 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH) was examined and compared with those of 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)-propionamide] (AMHP). The rate constant of free radical formation (ek(d)) for AIPH was 2.6 x 10(-6)/s at 37 degrees C in PBS (pH 7.4) solution, indicating that AIPH gives 3.8 times more free radicals than AAPH under the same conditions. It was found that the dynamics of oxidation and antioxidation induced by AIPH can be studied satisfactorily in the oxidation in micelles, LDL and erythrocyte suspensions, plasma, and cultured cells. The extent of cell death induced by AIPH and AAPH was directly proportional to the total free radicals formed. Interestingly, it was found that rats would not drink water containing AAPH, but they drank water containing AIPH. The levels of 8-iso-prostaglandin F2alpha (8-isoPs), 7-hydroxycholesterol (FCOH), lysophosphatidylcholine in the plasma of rats given water containing 50 mM AIPH for 1 month increased compared with those of control rats which drank water without AIPH. It may be concluded that AIPH is useful for kinetic and mechanistic studies on oxidative stress to membranes, lipoproteins, cells, and even animal models.