Role of quercetin and its in vivo metabolites in protecting H9c2 cells against oxidative stress.

The aim of this study was to investigate the potential of quercetin and two of its "in vivo" metabolites, 3'-O-methyl quercetin and 4'-O-methyl quercetin, to protect H9c2 cardiomyoblasts against H(2)O(2)-induced oxidative stress. As limited data are available regarding the potential uptake and cellular effects of quercetin and its metabolites in cardiac cells, we have evaluated the cellular association/uptake of the three compounds and their involvement in the modulation of two pro-survival signalling pathways: ERK1/2 signalling cascade and PI3K/Akt pathway. The three flavonols associated with cells to differing extents. Quercetin and its two O-methylated metabolites were able to reduce intracellular ROS production but only quercetin was able to counteract H(2)O(2) cell damage, as measured by MTT reduction assay, caspase-3 activity and DNA fragmentation assays. Furthermore, only quercetin was observed to modulate pro-survival signalling through ERK1/2 and PI3K/Akt pathway. In conclusion we have demonstrated that quercetin, but not its O-methylated metabolites, exerts protective effects against H(2)O(2) cardiotoxicity and that the mechanism of its action involves the modulation of PI3K/Akt and ERK1/2 signalling pathways.

Polyunsaturated fatty acids: From diet to binding to ppars and other nuclear receptors.

Dietary polyunsaturated fatty acids (PUFAs) function not only by altering membrane lipid composition, cellular metabolism, signal transduction, but possess also effects on gene expression by regulating the activity/abundance of different nuclear transcription factors: peroxisome proliferator activated receptors, retinoid X receptors, liver X receptors, hepatic nuclear factors-4a, and sterol regulatory binding proteins 1 and 2. PUFAs regulate the expression of genes in various tissues, including the liver, heart, adipose tissue, and brain, playing a major role in carbohydrate, fatty acid, triglyceride, and cholesterol metabolism. Before binding to transcription factors, PUFAs must be absorbed in the intestine and delivered to cells, and then they must enter the cell and the nucleus. PUFA concentration within the cell depends on many different factors, and regulate their possibility to act as transcription modulators. The aim of this review is to summarize recent knowledge about PUFAs destiny from dietto nuclear factors binding, examining the different variables which can modulate their interaction with nuclear factors themselves and therefore their effect on gene expression.

Green tea modulation of inducible nitric oxide synthase in hypoxic/reoxygenated cardiomyocytes.

Hypoxia/reoxygenation (H/R) is one of the causes of the increased expression of inducible nitric oxide synthase (iNOS) in cardiomyocytes. Since an aberrant NOS induction has detrimental consequences, we evaluated the effect of a green tea extract (GTE) on the NOS induction and activity in H/R-cardiomyocytes to define a nutritional strategy. Cultured rat cardiomyocytes were exposed to H/R in the presence of two concentrations of a green tea extract (GTE), which is reported to inhibit NOS expression and activity in different cells. In cultured cardiomyocytes two NOS isoforms were constitutively expressed, but only iNOS was induced by H/R. GTE supplementation at the lowest concentration, comparable to that in human plasma after dietary consumption, was ineffective, while the highest, comparable to that achievable by dietary supplements, counteracted the effect of H/R on iNOS induction and activity. It is necessary to verify in humans the relationship between the modulation of NO production and green tea dietary consumption.

Green tea extracts can counteract the modification of fatty acid composition induced by doxorubicin in cultured cardiomyocytes.

Doxorubicin cardiotoxicity is associated with the generation of free radicals, and involves not only lipid peroxidation but also a decreased biosynthesis of highly unsaturated fatty acids, leading to significant modification in cardiomyocyte fatty acid composition. We have evaluated whether naturally occurring antioxidants could counteract this side-effect. Green tea is an excellent source of catechins; we supplemented cultured rat cardiomyocytes with different green tea extracts to relate their catechin content and composition to their ability in protecting cells against doxorubicin-induced damage. The determination of total lipid fatty acid composition, of conjugated diene production (indicator of lipid peroxidation), and of lactate dehydrogenase release revealed that supplementation with tea extracts could counteract significant modifications in the fatty acyl pattern due to doxorubicin exposure, although to different extents. These differences could be ascribed to the different total catechin content and to qualitative differences among the tea extracts, determined by HPLC analysis.

Role of gamma-linolenic acid in counteracting doxorubicin-induced damage in cultured rat cardiomyocytes.

The clinical usefulness of doxorubicin is limited by cardiotoxicity. We have demonstrated that doxorubicin has a dual negative effect on myocardial lipids, acting against highly unsaturated fatty acids (HUFAs) directly and desaturating/elongating enzymes required for their biosynthesis, thus decreasing linoleic and alpha -linolenic conversion to higher metabolites. Primary cultures of rat cardiomyocytes were challenged with different doxorubicin concentrations and doxorubicin exposure was followed by a 24-h recovery period in the absence or presence of serum, and of gamma -linolenic acid. Serum in the recovery medium did not appear to be essential for the restoration of the desaturating/elongating activities, and gamma -linolenic acid supplementation influenced only alpha -linolenic acid conversion. Serum, and particularly gamma-linolenic acid, were very important in increasing HUFA levels behind the pure biosynthesis. HUFA biosynthesis plays a role in counteracting doxorubicin toxicity, but it cannot completely overcome the depletion of these fatty acids; serum and exogenous gamma-linolenate are critical in filling the decreased HUFA pool.

The protective role of different green tea extracts after oxidative damage is related to their catechin composition.

The antioxidant activities of three different green tea extracts were investigated and compared by two different methods. By the first method, which evaluated the direct protective effect of the green tea extracts on lipid peroxidation, the extracts were added, at different concentrations, to a lipid model system, made by refined peanut oil, freshly submitted to a further bleaching and subjected to forced oxidation at 98 degrees C, by an oxidative stability instrument. By the second method, the effectiveness of the same extracts was checked in cultures of neonatal rat cardiomyocytes exposed to a free radical-generating system by evaluating conjugated diene production and lactate dehydrogenase release. All of the extracts revealed a strong antioxidant activity by both the methods, and a particular effectiveness was demonstrated by the extracts having higher amounts of (-)-epigallocathechin-3-gallate and (-)-epigallocathechin, as analyzed by reverse-phase HPLC analysis.

Concentration- and time-dependent effects of gamma-linolenic acid supplementation to tumor cells in culture.

Gamma-linolenic acid (GLA) supplemented to neuroblastoma SK-N-BE, tubal carcinoma TG and colon carcinoma SW-620 cells was incorporated into phospholipids in all the cell lines (although to different extents), in a concentration- and time-dependent manner. All the cell lines were able to metabolize GLA to arachidonic acid, SK-N-BE being the most active. Supplementation with low GLA concentrations for short periods was not sufficient to impair cell proliferation; only higher amounts of GLA had an anti-proliferative effect also in short times. In these conditions, the antiproliferative effect of GLA is probably due to cellular dysfunction caused by fatty acid modifications.

Essential fatty acid metabolism in long term primary cultures of rat cardiomyocytes: a beneficial effect of n-6:n-3 fatty acids supplementation.

In long term (21 days) primary cultures of neonatal rat cardiomyocytes, utilized as a model of in vitro senescence, we investigated the dual effect of the time length in culture and of the supplementation with n-6:n-3 fatty acid mixtures on linoleic (LA) and alpha-linolenic acid (ALA) metabolism. Cardiomyocytes were divided into groups receiving: (1) control medium; (2) control medium plus n-3 fatty acids; (3) and (4) control medium plus n-6 and n-3 fatty acids in the ratio 1:2 or 2:1, respectively. In control cells. senescence caused a reduction in the conversion of LA and ALA, and the decrease in their metabolites was bypassed by the different supplementations. The fatty acid composition of cardiomyocyte lipids was therefore affected by both senescence and supplementation, as evidenced by the n-6:n-3 fatty acid ratio and the unsaturation index (U.I.) in cellular lipids. The final result of ageing in culture and of fatty acid supplementations was in all the groups of cells but one (n-6:n-3, 2:1) an unbalance in the n-6:n-3 fatty acid ratio. All the supplementations were able to counteract the decrease in the U.I. observed with senescence, but only the n-6:n-3 (2:1) was able to do so by increasing the cellular content of the fatty acids which are precursors of anti-aggregation eicosanoids without altering the n-6:n-3 fatty acid ratio.

Dual influence of aging and vitamin B6 deficiency on delta-6-desaturation of essential fatty acids in rat liver microsomes.

Delta-6-desaturase (D6D) activity is influenced by many nutritional and non-nutritional factors, among which one of the most important is aging. D6D activity could be susceptible to the dual influence of aging itself and of nutritional deficiencies, due to the reduced intake and/or absorption of essential nutrients. Particularly, vitamin B6 deficiency might be a crucial factor for D6D activity in aged people. Using 20 month old Sprague-Dawley rats fed a diet with a subnormal level of vitamin B6, we evaluated D6D activity for linoleic acid (LA) and alpha-linolenic acid (ALA) in liver microsomes, and the fatty acid composition of microsomal total lipids. We observed a diminished D6D activity for LA and also for ALA in vitamin B6-deficient animals, being approximately 63% and 81% respectively of the corresponding activity in control rats. As a consequence, significant modifications in the relative molar content of microsomal fatty acids were observed. The content of arachidonic and docosahexaenoic acid, the main products of the conversion of LA and ALA respectively, decreased, LA content increased and a decrease in the unsaturation index was observed in liver microsomes of B6-deficient rats. The foregoing results suggest that the impairment of D6D activity by vitamin B6 deficiency might be an important factor in decreasing the synthesis of n-6 and n-3 PUFAs. This may be particularly important in aging, where D6D activity is already impaired.

Manipulation of lipid composition of rat heart myocytes aged in culture and its effect on alpha1-adrenoceptor stimulation.

The fatty acid composition of the phosphoinositides was evaluated in cultured neonatal rat cardiomyocytes during the aging-like process in vitro, comparing data obtained from control and gamma-linolenic acid supplemented cardiomyocytes. The response to alpha1 stimulation was evaluated in both control and supplemented cells to verify the relationship between the alterations of the phosphoinositide fatty acid composition concomitant to culture aging and the cell response to exogenous stimuli. Arachidonate level decreased as a function of age in all the phosphoinositides, which appeared to be more saturated as cells aged in culture. Inositol phosphate production in response to alpha1 stimulation decreased as cells aged in culture. Supplementation of culture medium with gamma-linolenic acid caused significant modifications in the fatty acid pattern of the phosphoinositides, which appeared less saturated than the corresponding fractions isolated from unsupplemented cells during the aging-like process. The modifications induced by the supplementation in the phosphoinositide fatty acid composition prevented the age-related reduction of inositol phosphate production upon stimulation. These results clearly indicate a major role for the lipid composition in determining the response to alpha1 stimulation, suggesting a nutritional approach to overcome some of the impairments of molecular events related to the process of aging.

Age-related changes in essential fatty acid metabolism in cultured rat heart myocytes.

We previously demonstrated that cultured neonatal rat myocytes have the capacity to desaturate/elongate essential fatty acids, alpha-linolenic acid conversion being higher than linoleic acid conversion. The whole process of highly unsaturated fatty acid formation from linoleic and alpha-linolenic acids slows with aging. In this study we grew heart myocytes in culture for different periods of time, and we observed a decrease in the desaturating/elongating activities for both substrates as the cells aged in culture. Alpha-linolenic acid conversion into highly unsaturated fatty acids was less impaired by aging than linoleic acid conversion. These modifications are correlated to the age-dependent alterations observed in the total lipid fatty acid composition, which caused a decrease in the unsaturation index. Changes in the lipid composition that occur in aging cultures parallel those reported for several tissues upon aging in the whole animal. The data herein reported may suggest the possibility of counteracting the effects of aging on lipid metabolism by supplementing cultures with appropriate amounts of highly unsaturated fatty acids.

A randomized controlled study comparing elemental diet and steroid treatment in Crohn's disease.

BACKGROUND: Elemental diet is considered an effective primary treatment for active Crohn's disease, but it is usually given by a feeding tube. METHODS: Twenty-two patients (12 males, median age 30 years, range 18-60) with moderately active Crohn's disease were enrolled in a randomized study in which the efficacy of an elemental diet administered orally was compared to high-dose corticosteroids in achieving clinical and laboratory remission. Ten patients were treated by oral elemental diet (Peptamen, Clintec, USA) and 10 received corticosteroids. Both treatment regimens lasted 2 weeks. The two groups did not differ with respect to age, sex, body weight, location of disease, treatment or disease activity prior to the study. In all patients studied, simple Crohn's disease activity index, nutritional status (expressed as body mass index), percentage of ideal body weight, fat mass, fat free mass, erythrocyte sedimentation rate, interleukin-6, intestinal permeability (expressed as permeability index), prealbumin, retinol binding protein and multiskin test were evaluated before and after treatment. RESULTS: After 2 weeks of treatment, there were significant improvements in simple Crohn's disease activity index, erythrocyte sedimentation rate, permeability index, body mass index, prealbumin, retinol binding protein and multiskin test in the elemental diet group. There were significant improvements in simple Crohn's disease activity index and fat free mass in the corticosteroid group. CONCLUSIONS: These data suggest that, in the short term, an oral elemental diet is at least as effective as steroids in inducing remission of mild-moderately active Crohn's disease, but it may be more effective in improving the nutritional status of these patients, probably through a more rapid restoration of normal intestinal permeability.

Linoleic acid metabolism in primary cultures of adult rat cardiomyocytes is impaired by aging.

Many of the changes that occur in the rat cardiac muscle with advancing age are related to modifications in membrane fatty acid composition, polyunsaturated fatty acids decreasing and saturated increasing as the animal develops. In the present study, using cultured adult cardiomyocytes isolated from the hearts of rats of a broad (1-24 months) age range, we demonstrated that the modifications in the fatty acid pattern of cardiomyocytes have to be related to alterations in the mechanism of desaturation/elongation of essential fatty acids. In fact, independent of the age of the animal, heart cells in culture were capable of rapidly metabolizing radiolabeled linoleic acid taken up from the surrounding medium, but to a different extent. The ability of heart cells to metabolize linoleic acid to higher and more unsaturated metabolites decreased with the animal's age. As the age of the animal increased, the pattern of fatty acids of the cultured cardiomyocytes showed a gradual but significant shift, similar to those reported in the whole heart. Data here reported confirm that the basic aging-related process in the cellular model system may also be relevant to aging in the whole animal.

Essential fatty acid metabolism in cardiomyocytes grown in media enriched with different N-6/N-3 fatty acid combinations.

We have evaluated the effects of three different 18:3n-6, 20:5n-3 and 22:6n-3 fatty acid combinations on essential fatty acid (EFA) metabolism in rat cultured cardiomyocytes. The desaturating/elongating activities for linoleic (LA) and alpha-linolenic acid (ALA) were evaluated by radiolabeling the cells with 1-[14C]LA or 1-[14C]ALA and the fatty acid pattern of cardiomyocytes was assessed by gas chromatography. LA and ALA conversion to more unsaturated metabolites was reduced by increasing respectively n-3 and n-6 fatty acid concentration in the media. The all three combinations used reduced the saturated and increased the polyunsaturated fatty acid content of cardiomyocytes. The n-6/n-3 fatty acid ratio did not change compared to control cells in cardiomyocytes receiving the highest amount of 18:3n-6 and the lowest amounts of n-3 fatty acids. This combination may be suitable for modifying EFA desaturating/elongating activities without altering the physicochemical parameters which are related to the correct balance between n-6 and n-3 fatty acid content.

Visual evoked potentials and dietary long chain polyunsaturated fatty acids in preterm infants.

The influence of dietary long chain polyunsaturated fatty acid (LCP) supply, and especially of docosahexaenoic acid (DHA), on evoked potential maturation, was studied in 58 healthy preterm infants using flash visual evoked potentials (VEPs), flash electroretinography (ERG), and brainstem acoustic evoked potentials (BAEPs) at 52 weeks of postconceptional age. At the same time, the fatty acid composition of red blood cell membranes was examined. The infants were fed on breast milk (n = 12), a preterm formula supplemented with LCP (PF-LCP) (n = 21), or a traditional preterm formula (PF) (n = 25). In the breast milk and PF-LCP groups the morphology and latencies of the waves that reflect the visual projecting system were similar; in the PF group the morphology was quite different and the wave latencies were significantly longer. This could mean that the maturation pattern of VEPs in preterm infants who did not receive LCP was slower. Moreover, a higher level of erythrocyte LCP, especially DHA, was found in breast milk and PF-LCP groups compared with the PF group. ERG and BAEP recordings were the same in all three groups. These results suggest that a well balanced LCP supplement in preterm formulas can positively influence the maturation of visual evoked potentials in preterm infants when breast milk is not available.

Normalization of immune response and phosphoinositide fatty acid composition of peripheral blood lymphocytes in an alcoholic patient after alcohol abstinence.

After 10 months of alcohol abstinence a malnourished alcoholic patient improved his nutritional status. The analysis of peripheral blood lymphocyte response to mitogenic stimulation with the antibody anti-CD3 and of the fatty acid composition of the (poly)-phosphoinositide fraction derived from lymphocytes revealed: 1) a similar [3H]-thymidine uptake as in control (non-drinker) subjects; 2) a similar relative molar content of the main fatty acids in the (poly)-phosphoinositides as in control subjects. Alcohol abstinence can normalize both the parameters, which are greatly altered during alcohol abuse. This suggests a link between nutritional status and lymphocyte responsiveness via phosphoinositide fatty acid composition.

Defective calcium increase and inositol phosphate production in anti-CD3-stimulated lymphocytes of alcoholics without progressive liver disease.

Intracellular free calcium concentration, phosphoinositide turnover, and inositol phosphate production were analyzed in peripheral blood lymphocytes from seven well-nourished alcoholic patients without severe acute or chronic liver disease, before and after stimulation with anti-CD3 antibody. Seven comparable nondrinkers were studied as controls. A lower increase in intracellular free calcium concentration was detected in alcoholics, after anti-CD3 stimulation of lymphocytes, than in control subjects. Lymphocyte activation generated inositol phosphates in both controls and alcoholics, but inositol phosphate production was significantly lower in alcoholics. The agreement between these findings indicates that the reduction in inositol phosphates is one of the most important events in the early phases of lymphocyte activation in alcoholics.

Intestinal absorption of bile acids in the rabbit: different transport rates in jejunum and ileum.

BACKGROUND & AIMS: A direct comparison of jejunal and ileal absorption rates of bile acids has not been reported. The aim of this study was to compare the relative transport rates of different bile acids in the jejunum and ileum. METHODS: Jejunal and ileal rabbit intestinal segments were separately perfused with bile acid solutions, and dose-response curves were obtained for taurocholate, ursodeoxycholate, chenodeoxycholate, deoxycholate, and their glycoconjugates. Membrane fluidity and bile acid transport were assessed in brush border membrane vesicles. RESULTS: Taurocholate showed active transport in the ileum and no transport in the jejunum. Unconjugated bile acids showed passive diffusion in the two tracts, whereas glycoconjugated bile acids showed both components of transport in the ileum and passive diffusion in the jejunum (lower in the latter). A higher membrane fluidity and lower cholesterol-to-phospholipid ratio were found in the jejunum. Ursodeoxycholate reduced bile acid uptake into membrane vesicles from both ileum and jejunum. CONCLUSIONS: Active transport is limited to the ileum. Passive diffusion is higher through a less fluid membrane with a higher cholesterol-to-phospholipid ratio in the ileum than in the jejunum. Ursodeoxycholate inhibition may be at the level of a facilitated, sodium-independent diffusion in the jejunum.