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.

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.

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.

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.

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.

gamma-Linolenic acid supplementation can affect cancer cell proliferation via modification of fatty acid composition.

We examined the effect of gamma-linolenic acid (GLA) supplementation on the growth and fatty acid composition of three human tumor cell lines (the neuroblastoma CHP-212, the tubal carcinoma TG, and the colon carcinoma SW-620), in order to evaluate the relationship between GLA-induced tumor cell death and the distribution of fatty acids in tumor cells. At the highest GLA concentrations (10 and 20 micrograms/ml), the DNA synthesis was completely abolished; at 5 micrograms/ml GLA only SW-620 cells did not proliferate, while CHP-212 and TG cells showed a residual [3H]-thymidine incorporation. GLA levels were very low in cells grown in control medium; GLA supplementation caused a significant incorporation of GLA itself in all the cell lines at each concentration. In TG and CHP-212 cells, GLA was metabolized, although to a different extent, to dihomo-gamma linolenic acid and arachidonic acid. SW-620 cells neither elongated nor desaturated the incorporated GLA. The highest cytostatic effect was reached when GLA was not transformed into its metabolites, suggesting that the GLA toxicity to tumor cells is not dependent on metabolites but is due to GLA itself.

Metabolism of linoleic and alpha-linolenic acids in cultured cardiomyocytes: effect of different N-6 and N-3 fatty acid supplementation.

The metabolites of linoleic (LA) and alpha-linolenic (ALA) acids are involved in coronary heart disease. Both n-6 and n-3 essential fatty acids (EFAs) are likely to be important in prevention of atherosclerosis since the common risk factors are associated with their reduced 6-desaturation. We previously demonstrated the ability of heart tissue to desaturate LA. In this study we examined the ability of cultured cardiomyocytes to metabolize both LA and ALA in vivo, in the absence and in the presence of gamma linolenic acid (GLA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) alone or combined together. In control conditions, about 25% or LA and about 90% of ALA were converted in PUFAs. GLA supplementation had no influence on LA conversion to more unsaturated fatty acids, while the addition of n-3 fatty acids, alone or combined together, significantly decreased the formation of interconversion products from LA. Using the combination of n-6 and n-3 PUFAs, GLA seemed to counterbalance partially the inhibitory effect of EPA and DHA on LA desaturation/elongation. The conversion of ALA to more unsaturated metabolites was greatly affected by GLA supplementation. Each supplemented fatty acid was incorporated to a significant extent into cardiomyocyte lipids, as revealed by gas chromatographic analysis. The n-6/n-3 fatty acid ratio was greatly influenced by the different supplementations; the ratio in GLA+EPA+DHA supplemented cardiomyocytes was the most similar to that recorded in control cardiomyocytes. Since important risk factors for coronary disease may be associated with reduced 6-desaturation of the parent EFAs, administration of n-6 or n-3 EFA metabolites alone could cause undesirable effects. Since they appear to have different and synergistic roles, only combined treatment with both n-6 and n-3 metabolites is likely to achieve optimum results.

Essential fatty acid metabolism in cultured rat cardiomyocytes in response to either N-6 or N-3 fatty acid supplementation.

In this study we demonstrate that cultured rat cardiomyocytes possess the capacity to desaturate/elongate essential fatty acids (EFAs). Alpha-linolenic acid (ALA) conversion to higher metabolites was greater than linoleic acid (LA) conversion, according to the higher affinity of the delta-6-desaturase enzyme for the n-3 than for the n-6 EFAs. Gamma-linolenic acid (GLA) supplementation to the culture medium had no influence on LA conversion; but the addition of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids significantly decreased the formation of interconversion products from LA. The conversion of ALA to higher metabolites was greatly affected by GLA; EPA had no effect on ALA conversion, while DHA significantly inhibited it. Both GLA (converted mostly to dihomo-gamma-linolenic acid) and EPA can be removed from phospholipids and addressed to prostanoid biosynthesis, so avoiding their potential accumulation and the inhibition of their own production. Our data clearly indicate that supplementation of the culture medium with either n-6 or n-3 fatty acids can cause reduced levels of the other series of fatty acids. This effect may be undesirable, since both n-6 and n-3 fatty acids are important in the prevention of coronary diseases.

Incorporation of cholesterol oxidation products into cell lipids and their influence on the proliferation of cultured cardiomyocytes.

We have investigated the incorporation of cholesterol oxidation products into cardiomyocyte lipids and related this to changes in cell proliferation, evaluated by measuring cellular protein content. Primary cultures of neonatal rat ventricular cells were supplemented with scalar concentrations of several cholesterol oxidation products (cholestan-5 alpha, 6 alpha-epoxy-3 beta-ol, 5 alpha-cholestane-3 beta, 5, 6 beta-triol, 5-cholesten-3 beta, 4 beta-diol, 5-cholesten-3 beta-ol-7-one, and 5-cholesten-3-one). Although all the cholesterol oxidation products were incorporated into the cardiomyocyte lipids when added to the medium at a concentration higher than 0.5 microM, the extent of the incorporation of the different cholesterol oxidation products differed, depending on the concentration in the culture medium and on the chemical structure of the compound. The effects of the cholesterol oxidation products on the cellular protein content were also different: 5 alpha-cholestane-3 beta, 5, 6 beta-triol was shown to be the most potent inhibitor of cell proliferation, followed by cholestan-5 alpha, 6 alpha-epoxy-3 beta-ol, 5-cholesten-3 beta, 4 beta-diol and 5-cholesten-3 beta-ol-7-one. 5-Cholesten-3-one did not affect the cellular protein content. The ability of cholesterol oxidation products to inhibit cell proliferation, and their capacity to increase the permeability of the plasma membrane to calcium, could be deleterious for cardiac cells.

The effect of gamma-linolenic acid on clinical status, red cell fatty acid composition and membrane microviscosity in infants with atopic dermatitis.

A double blind placebo-controlled study of two doses of gamma-linolenic acid, provided by evening primrose oil (EPO, Epogam, Searle, U.K.), in children with atopic dermatitis was performed: 1) to examine the effect of gamma-linolenic acid administration on the clinical status of children with atopic dermatitis and abnormalities of IgE-mediated immune responses compared to those without such IgE abnormalities; 2) to investigate the effect of gamma-linolenic acid on red cell fatty acid composition and 3) to assess whether treatment with gamma-linolenic acid induced changes in red cell membrane microviscosity. A significant improvement in the overall severity of the clinical condition was seen in children treated with gamma-linolenic acid, independent of whether the children had manifestations of IgE-mediated allergy. Furthermore, gamma-linolenic acid treatment increased the percentage content of n-6 fatty acids in erythrocyte cell membrane; this increase was more marked in the membranes of children treated with high doses of EPO. In the high dose group a significant increase in dihomogamma-linolenic acid (DGLA) occurred. This may be of particular relevance because of the potential importance of DGLA as a precursor of antiinflammatory prostanoids. Red cell membrane microviscosity did not change in any group after treatment with EPO, even in high doses, despite a significant increase in the proportion of long chain polyunsaturated fatty acids.

[Tertiary hyperparathyroidism during chronic kidney failure under dialysis treatment. Apropos a clinical case]. / Iperparatiroidismo terziario in corso di insufficienza renale cronica in trattamento dialitico. Considerazioni a proposito di un caso clinico.

The authors describes a case of tertiary hyperparathyroidism (HPTH) in a uremic patient on intermittent dialysis treatment: the term refers to an adenoma with ensuing uncontrolled parathormone (PTH) secretion rate arising on the ground of hyperstimulated hypertrophied parathyroid glands. The syndrome was heralded clinically by bone pain, psychiatric disorder and biochemically by increased levels of calcium and alkaline phosphatase (AP), while parathormone (PTH), did not change from basal very high levels as commonly found in uraemic patients. As hypercalcemia in the hemodialyzed is an infrequent finding the only alternative explanation could have been hypercalcemic secondary HPTH related to hyperplastic autonomous parathyroids. For no clinical and laboratory findings as well as US findings and double scintigraphy (99mTc and 201mTl) may suggest differential diagnosis the patient underwent total parathyroidectomy which actually revealed an adenoma of the left superior parathyroid gland. Bone pain and psychiatric disturbances disappeared and now get well on chronic dialysis treatment and 1.25-OH Vit D3 supplement.

Diacylglycerol fatty acid composition is related to activation of protein kinase C in cultured cardiomyocytes.

Using cultures of beating neonatal rat cardiomyocytes we have studied the fatty acid composition of the diacylglycerol produced after different stimulation times with an alpha 1-agonist (phenylephrine) and we have related it to the previously reported time course of the activation of particulate protein kinase C, in control cells and in cells grown in a medium supplemented with docosahexaenoic acid. Gas chromatography of the diacylglycerol produced after stimulation revealed significant differences between control cells and cells treated with docosahexanoic acid. In the cells treated with docosahexanoic acid, the more persistent activation of the membrane-bound protein kinase C might be sustained by an enrichment of diacylglycerol with docosahexanoic acid. The modification of the fatty acid composition of diacylglycerol can cause an alteration in the response of the cells to alpha 1-adrenoceptor stimulation.

[The correlation between the acidic composition of diacylglycerol and protein kinase C activation in cultures of rat cardiomyocytes]. / Correlazione tra composizione acidica del diacilglicerolo e attivazione della proteina cinasi C in colture di cardiomiociti di ratto.

We have studied the fatty acid composition of the diacylglycerol produced after different stimulation times with an alpha 1-agonist (phenylephrine) in cultures of beating neonatal rat cardiomyocytes, and we have related the acidic pattern to the time course of the translocation of protein kinase C from cytosol to the membrane, both in control cells and in cells grown in a medium supplemented with docosahexaenoic acid. Gas chromatography of the diacylglycerol produced after stimulation revealed significant differences between control cells and cells grown in the docosahexaenoic acid supplemented medium. In the control cells, in the early stimulation times, the higher protein kinase C activity was due to a higher relative molar content of arachidonic acid in the diacylglycerol; in the docosahexaenoic acid treated cells the lower but more persistent activation of the membrane-bound protein kinase C might be sustained by an enrichment of diacylglycerol with docosahexaenoic acid. The modification of the fatty acid composition of diacylglycerol can cause an alteration in the response of the cells to alpha 1-adrenoceptor stimulation.

Fatty acid composition of phosphoinositides in cultured cardiomyocytes: effects of docosahexaenoic acid and alpha 1-adrenoceptor stimulation.

Using cultures of beating cardiomyocytes from neonatal rats, we have studied the fatty acid composition of phosphatidylinositol, phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate and the effect of supplementing the culture medium with docosahexaenoic acid on the fatty acid composition of the three phosphoinositides. Docosahexaenoic acid was incorporated into the phosphatidylinositol fraction of the supplemented cells, but not into the phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate fractions. At complete confluence, the cardiomyocytes were stimulated with an alpha 1-agonist (phenylephrine). This altered the acidic pattern of the phosphoinositides in both control and supplemented cells. The differences observed between the polyphosphorylated classes and the phosphatidylinositol fraction suggest the existence of different mechanisms of selection of fatty acids in the biosynthesis of phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate.

Protein kinase C activity in neonatal cultured rat cardiomyocytes supplemented with docosahexaenoic acid.

In vitro studies have indicated that the 1-stearoyl, 2-arachidonyl diacylglycerol (DAG) is the most effective one for the activation of protein kinase C, although many other DAGs having a different fatty acid composition are active, but to a different extent. Using cultures of neonatal rat ventricular cells, grown in a medium enriched in docosahexaenoic acid (DHA), we previously obtained a cell population that, after alpha 1-adrenoceptor stimulation, produced a DHA enriched DAG. In this study, we have tested the "in vivo" ability of this modified DAG as protein kinase C activator, demonstrating a lower but more persistent translocation of the enzyme from cytosol to particulate fraction in the DHA treated cells. The differences in the PKC activation pattern could be explained by a different metabolism of the DHA enriched DAG by DAG kinase.