An encapsulated fruit and vegetable juice concentrate increases skin microcirculation in healthy women.

BACKGROUND/AIM: Microcirculation in the dermis of the skin is important for nutrient delivery to this tissue. In this study, the effects of a micronutrient concentrate (Juice Plus+®; 'active group'), composed primarily of fruit and vegetable juice powder, on skin microcirculation and structure were compared to placebo. STUDY DESIGN/METHODS: This 12-week study had a monocentric, double-blind placebo and randomized controlled design with two treatment groups consisting of 26 healthy middle-aged women each. The 'oxygen to see' device was used to evaluate microcirculation. Skin density and thickness were measured using ultrasound. Measurements for skin hydration (Corneometer®), transepidermal water loss and serum analysis for carotenoids and α-tocopherol were also performed. RESULTS: By 12 weeks, microcirculation of the superficial plexus increased by 39%. Furthermore, skin hydration increased by 9% while skin thickness increased by 6% and skin density by 16% in the active group. In the placebo group, microcirculation decreased, and a slight increase in skin density was observed. CONCLUSION: Ingestion of a fruit- and vegetable-based concentrate increases microcirculation of the skin at 12 weeks of intervention and positively affects skin hydration, density and thickness.

Plasma levels of vitamin E and carotenoids are decreased in patients with Nonalcoholic Steatohepatitis (NASH).

OBJECTIVES: Oxidative stress is suggested to play an important role in the pathogenesis of nonalcoholic steatohepatitis (NASH). The present study was aimed to compare plasma levels of antioxidants in patients suffering from NASH and healthy controls. METHODS: Plasma levels of the antioxidants α-tocopherol, γ-tocopherol, lutein, zeaxanthin, ß-cryptoxanthin, lycopene, α-carotene ß-carotene were determined in 57 patients with biopsy-proven NASH and 40 healthy controls. RESULTS: Levels of α-tocopherol (22.4 vs. 26.8 nmol/ ml; p<0.01), lutein (0.19 vs. 0.33 nmol/ml; p<0.0001), zeaxanthin (0.04 vs. 0.08 nmol/ml; p<0.0001), lyco?pene (0.15 vs. 0.42 nmol/ml; p<0.0001), α-carotene (0.03 vs. 0.06 nmol/ml; p<0.005) and ß-carotene (0.25 vs. 0.39 nmol/ml; p<0.01) were significantly decreased in NASH patients compared to controls. Age, aminotransferase status (ALT, AST) and BMI were not correlated with the levels of tocopherols or caro?tenoids. CONCLUSIONS: Given the decreased levels supplementation of lipophilic antioxidants might be a rational treatment option for patients with NASH.

Anticoccidial and antiinflammatory activity of garlic in murine Eimeria papillata infections.

Coccidiosis with the protozoan parasite Eimeria as the infectious agent causes enormous economic losses, particularly in poultry farms. Here, we investigated the effects of garlic on the outcome of coccidiosis caused by Eimeria papillata in male Balb/c mice. The data showed that mice infected with E. papillata revealed an output of 3260 ± 680 oocysts per gram faeces on day 4 p.i.. This output is significantly decreased to 1820 ± 415 oocysts in garlic-treated mice. Infection also induced inflammation and injury of the liver. This was evidenced (i) as increases in inflammatory cellular infiltrations, dilated sinusoids, and vacuolated hepatocytes, (ii) as increased mRNA levels of inducible nitric oxide synthase (iNOS) and of the cytokines interferon gamma (IFN-γ), and interleukin-6 (IL-6), (iii) as increased plasma levels of alanine and aspartate aminotransferases, alkaline phosphatase, γ-glutamyl transferase and total bilirubin, (iv) as increased production of nitric oxide derived products (nitrite/nitrate) and malondialdehyde, and (v) as lowered glutathione levels and decreased activities of catalase and superoxide dismutase, respectively. All these infection-induced parameters were significantly less altered during garlic treatment. In particular, garlic counteracted the E. papillata-induced loss of glutathione and the activities of catalase and superoxide dismutase. Our data indicated that garlic treatment significantly attenuated inflammation and injury of the liver induced by E. papillata infections.

Opposite roles of selenium-dependent glutathione peroxidase-1 in superoxide generator diquat- and peroxynitrite-induced apoptosis and signaling.

Oxidative injuries including apoptosis can be induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) in aerobic metabolism. We determined impacts of a selenium-dependent glutathione peroxidase-1 (GPX1) on apoptosis induced by diquat (DQ), a ROS (superoxide) generator, and peroxynitrite (PN), a potent RNS. Hepatocytes were isolated from GPX1 knockout (GPX1-/-) or wild-type (WT) mice, and treated with 0.5 mm DQ or 0.1-0.8 mm PN for up to 12 h. Loss of cell viability, high levels of apoptotic cells, and severe DNA fragmentation were produced by DQ in only GPX1-/- cells and by PN in only WT cells. These two groups of cells shared similar cytochrome c release, caspase-3 activation, and p21(WAF1/CIP1) cleavage. Higher levels of protein nitration were induced by PN in WT than GPX1-/- cells. Much less and/or slower cellular GSH depletion was caused by DQ or PN in GPX1-/- than in WT cells, and corresponding GSSG accumulation occurred only in the latter. In conclusion, it is most striking that, although GPX1 protects against apoptosis induced by superoxide-generator DQ, the enzyme actually promotes apoptosis induced by PN in murine hepatocytes. Indeed, GSH is a physiological substrate for GPX1 in coping with ROS in these cells.

Enhanced gamma-glutamyl transpeptidase expression and superoxide production in Mpv17-/- glomerulosclerosis mice.

Recently, gamma-glutamyl transpeptidase, which initiates cleavage of extracellular glutathione, has been shown to promote oxidative damage to cells. Here we examined a murine disease model of glomerulosclerosis, involving loss of the Mpv17 gene coding for a peroxisomal protein. In Mpv17-/- cells, enzyme activity and mRNA expression (examined by quantitative RT-PCR) of membrane-bound gamma-glutamyl transpeptidase were increased, while plasma glutathione peroxidase and superoxide dismutase levels were lowered. Superoxide anion production in these cells was increased as documented by electron spin resonance spectroscopy. In the presence of Mn(III)tetrakis(4-benzoic acid)porphyrin, the activities of gamma-glutamyl transpeptidase and plasma glutathione peroxidase were unchanged, suggesting a relationship between enzyme expression and the amount of reactive oxygen species. Inhibition of gamma-glutamyl transpeptidase by acivicin reverted the lowered plasma glutathione peroxidase and superoxide dismutase activities, indicating reciprocal control of gene expression for these enzymes.

Dietary tomato paste protects against ultraviolet light-induced erythema in humans.

Carotenoids are efficient antioxidants capable of scavenging reactive oxygen species generated under conditions of photooxidative stress. It has been shown that supplementation with high doses of beta-carotene protects skin against UV-induced erythema. This study was designed to investigate whether intervention with a natural dietary source rich in lycopene protects against UV-induced erythema in humans. Tomato paste (40 g), providing approximately 16 mg/d of lycopene, was ingested with 10 g of olive oil over a period of 10 wk by 9 volunteers. Controls (n = 10) received olive oil only. Erythema was induced by illumination of dorsal skin (scapular region) with a solar simulator at the beginning of the study, after 4 wk and after 10 wk. Intensity of erythema was measured by chromatometry; the a-value was determined directly before and 24 h after irradiation. Serum carotenoid levels were measured by HPLC. At the beginning of the study, carotenoid levels did not differ between the two groups. Serum levels of lycopene increased in supplemented subjects; the other carotenoids did not change significantly, and no change in serum carotenoids was observed in the control group. At wk 10, dorsal erythema formation was 40% lower in the group that consumed tomato paste compared with controls (P = 0.02; Wilcoxon-Mann-Whitney test). No significant difference between groups was found at wk 4 of treatment. The data demonstrate that it is feasible to achieve protection against UV light-induced erythema by ingestion of a commonly consumed dietary source of lycopene.

Polyphenols of cocoa: inhibition of mammalian 15-lipoxygenase.

Some cocoas and chocolates are rich in (-)-epicatechin and its related oligomers, the procyanidins. Fractions of these compounds, isolated from the seeds of Theobroma cacao, caused dose-dependent inhibition of isolated rabbit 15-lipoxygenase-1 with the larger oligomers being more active; the decamer fraction revealed an IC50 of 0.8 microM. Among the monomeric flavanols, epigallocatechin gallate (IC50 = 4 microM) and epicatechin gallate (5 microM) were more potent than (-)-epicatechin (IC50 = 60 microM). (-)-Epicatechin and procyanidin nonamer also inhibited the formation of 15-hydroxy-eicosatetraenoic acid from arachidonic acid in rabbit smooth muscle cells transfected with human 15-lipoxygenase-1. In contrast, inhibition of the lipoxygenase pathway in J774A.1 cells transfected with porcine leukocyte-type 12-lipoxygenase (another representative of the 12/15-lipoxygenase family) was only observed upon sonication of the cells, suggesting a membrane barrier for flavanols in these cells. Moreover, epicatechin (IC50 approx. 15 microM) and the procyanidin decamer inhibited recombinant human platelet 12-lipoxygenase. These observations suggest general lipoxygenase-inhibitory potency of flavanols and procyanidins that may contribute to their putative beneficial effects on the cardiovascular system in man. Thus, they may provide a plausible explanation for recent literature reports indicating that procyanidins decrease the leukotriene/prostacyclin ratio in humans and human aortic endothelial cells.

Thyroid hormone receptors bind to an element in the connexin43 promoter.

Thyroid hormones stimulate gap junctional communication in rat liver WB-F344 epithelial cells, elevating connexin43 mRNA and protein levels. In the present work we analysed connexin43 expression in liver and heart samples from thyroid hormone-treated Wistar rats. Connexin43 mRNA was elevated 2.1-fold in rat liver samples as compared to controls, while there was no change in heart. Thyroid hormone response elements in the rat connexin43 promoter region were examined; a candidate sequence, including a binding site for ligand-dependent transcription factors, was identified at position -480 to -464. This putative regulatory element, rCx-480, contains a direct repeat structure separated by three base pairs (DR3-type element). In electrophoretic mobility shift assays using in vitro translated proteins, the rCx-480 element formed stronger complexes with thyroid hormone receptor alpha/retinoid X receptor alpha heterodimers than with vitamin D receptor/retinoid X receptor alpha heterodimers. In transfected Cos-7 cells, promoter activation was observed via this element after treatment with 3,3',5-triiodo-L-thyronine. Loss of binding was seen when the 3' half-site or the spacer region of the rCx-480 element were experimentally mutated, while a stronger binding was observed with mutations introduced in the 5' half-site.

Carotenoids and carotenoids plus vitamin E protect against ultraviolet light-induced erythema in humans.

BACKGROUND: Carotenoids and tocopherols, known to be efficient antioxidants and capable of scavenging reactive oxygen species generated during photooxidative stress, may protect the skin from ultraviolet light-induced erythema. beta-Carotene is widely used as an oral sun protectant but studies on its protective effects are scarce. OBJECTIVE: The objective of this study was to investigate the protective effects of oral supplementation with carotenoids and a combination of carotenoids and vitamin E against the development of erythema in humans. DESIGN: A carotenoid supplement (25 mg total carotenoids/d) and a combination of the carotenoid supplement and vitamin E [335 mg (500 IU) RRR-alpha-tocopherol/d] were given for 12 wk to healthy volunteers. Erythema was induced by illumination with a blue-light solar simulator. Serum beta-carotene and alpha-tocopherol concentrations and skin carotenoid levels were assessed by HPLC and reflection photometry. RESULTS: Serum beta-carotene and alpha-tocopherol concentrations increased with supplementation. Erythema on dorsal skin (back) was significantly diminished (P < 0.01) after week 8, and erythema suppression was greater with the combination of carotenoids and vitamin E than with carotenoids alone. CONCLUSION: The antioxidants used in this study provided protection against erythema in humans and may be useful for diminishing sensitivity to ultraviolet light.

Plasma levels of lipophilic antioxidants in very old patients with type 2 diabetes.

BACKGROUND: Experimental research indicates that oxidative stress is implicated in aging and in the pathogenesis of diabetes and its complications. This evidence is limited in elderly patients with non-insulin dependent diabetes, in which age- and disease-related production of reactive oxygen species might exert synergistic damaging effects on tissues and organs. METHODS: Plasma levels of lipid-soluble compounds with antioxidant properties including vitamin A, vitamin E and carotenoids (lutein, zeaxanthin, beta-cryptoxanthin, lycopene, alpha- and beta-carotene) were measured by HPLC in 72 elderly patients with non-insulin dependent diabetes (75.7+/-0.8 years, 40 F, 32 M) and in 75 age-matched controls (77.2+/-1.2 years, 48 F, 27 M). RESULTS: All compounds measured were significantly lower in plasma from diabetic patients as compared to controls (p<0.0001). Plasma levels of vitamins A and E and of carotenoids did not significantly correlate with dietary intake and lipid profile in both groups. In patients, significant inverse correlations were found between age and levels of vitamin E, beta-cryptoxanthin, lycopene and beta-carotene. CONCLUSIONS: We conclude that patients of very old age with Type 2 diabetes show a poor plasma status of vitamins A and E and carotenoids, which negatively correlates with age. Further studies are needed to explore the possible therapeutic role of lipid-soluble vitamin supplements in elderly diabetic subjects.

Quantification of the alpha- and gamma-tocopherol metabolites 2,5,7, 8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman and 2,7, 8-trimethyl-2-(2'-carboxyethyl)-6-hydroxychroman in human serum.

alpha- and gamma-tocopherol are the major vitamin E compounds found in human blood and tissues. The metabolites are 2,5,7, 8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC) and 2,7,8-trimethyl-2-(2'-carboxyethyl)-6-hydroxychroman (gamma-CEHC, LLU-alpha), respectively. alpha-CEHC is excreted mainly as glucuronide or sulfate conjugates in the urine. Here we describe a sensitive and reliable method to analyze alpha- and gamma-CEHC in human serum. The concentration of alpha-CEHC in human serum is in the range of 5-10 pmol/ml but increases significantly up to 200 pmol/ml upon supplementation with RRR-alpha-tocopherol. About one-third of the alpha-CEHC circulating in the blood is present as a glucuronide conjugate. Baseline levels of gamma-CEHC are about 50 to 85 pmol/ml.

Activation pattern of mitogen-activated protein kinases elicited by peroxynitrite: attenuation by selenite supplementation.

Peroxynitrite is a mediator of toxicity in pathological processes in vivo and causes damage by oxidation and nitration reactions. Here, we report a differential induction of mitogen-activated protein kinases (MAPKs) in WB-F344 rat liver epithelial cells by peroxynitrite. For the exposure of cultured cells with peroxynitrite, we employed a newly developed infusion method. At 6.5 microM steady-state concentration, the activation of p38 MAPK was immediate, while JNK1/2 and ERK1/2 were activated 60 min and 15 min subsequent to 3 min of exposure to peroxynitrite, respectively. Protein-bound 3-nitrotyrosine was detected. When cells were grown in a medium supplemented with sodium selenite (1 microM) for 48 h, complete protection was afforded against the activation of p38 and against nitration of tyrosine residues. These data suggest a new role for peroxynitrite in activating signal transduction pathways capable of modulating gene expression. Further, the abolition of the effects of peroxynitrite by selenite supplementation suggests a protective role of selenium-containing proteins.

Peroxynitrite diminishes gap junctional communication: protection by selenite supplementation.

Loss of intercellular communication via gap junctions has been correlated with progression of cells to a malignant phenotype. Here, we show that peroxynitrite, a mediator of toxicity in inflammatory processes, diminishes gap junctional intercellular communication (GJIC) in WB-F344 rat liver epithelial cells, assayed by the scrapeloading dye-transfer technique as well as by microinjection of a fluorescent dye into single cells. Exposure of cultured cells to a steady-state concentration of peroxynitrite of 1.6 microM for 4 min or to 3-morpholinosydnonimine (SIN-1) at 0.5 mM strongly diminished GJIC. These concentrations of peroxynitrite or SIN-1 were not cytotoxic. When cells were grown in a medium supplemented with sodium selenite (0.1-1 microM) for 72 h, substantial protection was afforded against the decrease in GJIC by peroxynitrite. Thus, peroxynitrite can disrupt GJIC, and selenium-containing proteins protect.

Increased dermal carotenoid levels assessed by noninvasive reflection spectrophotometry correlate with serum levels in women ingesting Betatene.

beta-Carotene is being used as an oral sun protectant, and evidence indicates that carotenoids may protect human skin from light-induced lesions. However, limited information is available on the distribution and accumulation of beta-carotene in skin, especially with respect to various skin regions. With the use of reflection spectroscopy, we investigated the accumulation of total carotenoids in human skin after repeated supplementation of 12 women with beta-carotene from a natural source Betatene, an algal extract. After daily ingestion of 24 mg beta-carotene (in Betatene) for 12 wk, an increase in carotenoid skin levels was observed. Highest basal values were measured in skin of the forehead, palm of the hand and dorsal skin, with lower levels measured in skin of the arm and back of the hand. Upon treatment, increases in carotenoid skin levels were found in all areas as follows: 2.4-fold in forehead, 0.7-fold in dorsal skin, 2.2-fold in the palm of the hand, 17-fold on the back of the hand and 1.7-fold on the inside of the arm. After cessation of treatment, the carotenoid levels decreased in all skin areas. Serum beta-carotene levels were elevated upon treatment and correlated with carotenoid skin levels. Correlations for serum vs. skin from the palm of the hand (r = 0.94) and skin from the forehead (r = 0.89) were calculated, indicating that serum levels appeared to be a suitable indicator for carotenoid accumulation in specific regions of the skin. With doses of approximately 20-25 mg carotenoids/d, it is possible to raise dermal carotenoid levels.

Evidence that singlet oxygen-induced human T helper cell apoptosis is the basic mechanism of ultraviolet-A radiation phototherapy.

Ultraviolet A (UVA) irradiation is effectively used to treat patients with atopic dermatitis and other T cell mediated, inflammatory skin diseases. In the present study, successful phototherapy of atopic dermatitis was found to result from UVA radiation-induced apoptosis in skin-infiltrating T helper cells, leading to T cell depletion from eczematous skin. In vitro, UVA radiation-induced human T helper cell apoptosis was mediated through the FAS/FAS-ligand system, which was activated in irradiated T cells as a consequence of singlet oxygen generation. These studies demonstrate that singlet oxygen is a potent trigger for the induction of human T cell apoptosis. They also identify singlet oxygen generation as a fundamental mechanism of action operative in phototherapy.

Antioxidant vitamins in prevention.

The authors are aware that there is still a need for much research to elucidate the possible preventive effects of antioxidant vitamins with regard to degenerative and neoplastic diseases. There must also be vigorous examination of the evidence that antioxidant vitamins can play a crucial part in a large number of other disorders (Alzheimer's disease, Parkinson's disease, diabetes, chronic and acute inflammations, airway disorders, reperfusion syndrome). The aim of prevention-oriented medical research must be to develop suitable measures able to make a considerable contribution to the overall prevention policy. Although there is still uncertainty about the mode of action and the optimal dosage of antioxidant nutrients and dietary constituents, particularly because of the safety when the dosage is correct, more information must be provided about early prevention of an inappropriate, low antioxidant intake; intake in the diet should definitely be preferred to supplementation because of the other beneficial effects of the recommended foodstuffs.

Vitamin E in humans: demand and delivery.

How much vitamin E is enough? An established use of supplemental vitamin E in humans is in the prevention and therapy of deficiency symptoms. The cause of vitamin E deficiency, characterized by peripheral neuropathy and ataxia, is usually malabsorption-a result of fat malabsorption or genetic abnormalities in lipoprotein metabolism. Genetic abnormalities in the hepatic alpha-tocopherol transfer protein also cause vitamin E deficiency-defects in this protein cause an impairment in plasma vitamin E transport. Impaired delivery of vitamin E to tissues, thereby, results in deficiency symptoms. Also discussed is the use of supplemental vitamin E in chronic diseases such as ischemic heart disease, atherosclerosis, diabetes, cataracts, Parkinson's disease, Alzheimer's disease, and impared immune function, as well as in subjects receiving total parenterol nutrition. In healthy individuals, a daily intake of about 15-30 mg of alpha-tocopherol is recommended to obtain "optimal plasma alpha-tocopherol concentrations" (30 microM or greater).

Human serum concentrations of all-trans beta- and alpha-carotene but not 9-cis beta-carotene increase upon ingestion of a natural isomer mixture obtained from Dunaliella salina (Betatene).

The uptake of all-trans and 9-cis beta-carotene and of alpha-carotene from a natural carotene preparation from Dunaliella salina, Betatene, was studied in humans. All-trans beta-carotene and alpha-carotene were absorbed well and showed the expected biokinetics with serum peak concentrations between 24 and 48 h. The mean increase in serum concentrations of alpha-carotene was 5.6% of the increase of all-trans beta-carotene, reflecting the composition of these carotenoids in Betatene. 9-cis beta-Carotene, however, was not detected in human serum, even after repeated doses. This could be due to preferential absorption of all-trans beta-carotene, rapid distribution of 9-cis beta-carotene into the tissue, or the presence of isomerase activity processing 9-cis to all-trans beta-carotene.

Reactivity of ebselen and related selenoorganic compounds with 1,2-dichloroethane radical cations and halogenated peroxyl radicals.

The reactivity of ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)one, and structurally related analogues was studied by pulse radiolysis. The rate constant for the reaction of ebselen with trichloromethylperoxyl radicals was determined to be 2.9 X 10(8) M-1 s-1, while its sulfur analogue, 2-phenyl-1,2-benzisothiazol-3(2H)one, was oxidized at much lower rates, k less than or equal to 10(7) M-1 s-1. Among several derivatives studied, the only other compound that exhibited a high rate constant was 2-(methylseleno)-benzoic acid-N-phenylamide. Oxidation of ebselen by other halogenated peroxyl radicals was also carried out and revealed a direct relationship between rate constant and the degree of halogenation of the oxidant. The transient radicals generated during oxidation of ebselen and the analogues were characterized by optical absorption and conductivity measurements and were attributed to one-electron-oxidized radical cations. The oxidation potentials were determined by cyclic voltammetry. Comparative evaluation of the in vitro behavior during microsomal lipid peroxidation revealed ebselen to be the most potent antioxidant of the compounds investigated, 2-(Methylseleno)-benzoic acid-N-phenylamide, despite its high rate constant for oxidation by halogenated peroxyl radicals, was found to be a poor antioxidant. The rate constant of oxidation of ebselen by trichloromethylperoxyl radicals is comparable to that of alpha-tocopherol under similar conditions, underscoring the potential pharmacological interest of ebselen as an antioxidant.

Oxidative damage to mitochondria and protection by ebselen and other antioxidants.

Iron/ascorbate induced lipid peroxidation in liver mitochondria isolated from normal and glutathione-depleted rats was monitored by low-level chemiluminescence and by accumulation of thiobarbituric acid-reactive substances (TBARS). Antioxidant capacity was assessed by the duration of the lag phase preceding the onset of active peroxidation. The lag phases in state 4 and in the presence of uncouplers were similar, but shorter in the presence of ADP (state 3). In glutathione-depleted rats the lag periods were less than those in normal mitochondria. A biphasic pattern of loss of membrane alpha-tocopherol was typical in state 4 with about 55% remaining after 40 min, while in presence of ADP there was a steady and rapid loss to about 30% of the initial level. Synthetic antioxidants such as ebselen or its glutathione adduct protected mitochondrial membranes against peroxidative reactions. There was a 5-fold increase in the lag phase with 1 microM ebselen in state 4 (lag doubling concentration, 0.4 microM) and a significantly lower rate of loss of alpha-tocopherol with about 90% of the initial level still remaining after 40 min. Likewise, the lag doubling concentrations were 0.04 microM for diethyldithiocarbamate, 0.3 microM for 5-hydroxyindole, 10 microM for dihydroxyphenylalanine and serotonin, and about 40 microM for epinephrine and norepinephrine.