Antioxidant activity of extracts from plants growing in Sardinia.

The present work was carried out to study the effect of some plant methanol extracts and essential oils on lipid peroxidation in simple in vitro systems. The tested extracts were obtained from four plants, commonly known in the Mediterranean area, indigenous to Sardinia: Artemisia arborescens L., Calycotome villosa L., Daphne gnidium L. or naturalized in the island, Eucalyptus globulus Labill. The activity of the extracts was investigated during both autoxidation and iron or EDTA-mediated oxidation of linoleic acid at 37 degrees C in the absence of solvent, and compared with that of BHT, alpha-tocopherol and EDTA. During linoleic acid autoxidation all the extracts were active, showing an antioxidant activity in the order: BHT >alpha- tocopherol >Daphne gnidium (methanol extract) >Eucalyptus globulus (essential oil) >Calycotome villosa (essential oil) >Artemisia arborescens (essential oil and methanol extract) >Calycotome villosa (methanol extract). None showed any prooxidant activity. During the iron-catalysed oxidation of linoleic acid the oils were not active, while all the methanol extracts showed some efficiency in preventing the oxidation process. All the extracts were also tested on cell cultures to investigate their cytotoxic activity or their ability to inhibit the growth of some pathogenic microorganisms.

Changes in conjugated linoleic acid and its metabolites in patients with chronic renal failure.

BACKGROUND: Conjugated linoleic acid (CLA) is a mixture of isomers of linoleic acid with conjugated double bonds that constitutes the most abundant fatty acid with conjugated dienes (CDs) in humans. CLA, erroneously considered in the past as a product of lipoperoxidation, has a dietary origin and has shown to possess anticarcinogenic and anti-atherogenic activity, mainly in animal studies. CLA can be metabolized to conjugated linolenic acid (CD18:3) and to conjugated eicosatrienoic acid (CD20:3) and these metabolites may be implicated in CLA activity. Because of the presence of dyslipidemia and the high incidence of cardiovascular and neoplastic diseases in uremic patients, we evaluated CLA and its metabolites in these patients in order to evaluate their metabolism and site distribution. METHODS: We measured CLA, CD18:3, CD20:3, CD fatty acid hydroperoxides (lipoperoxidation products), and linoleic acid in the plasma, adipose tissue, and red blood cell (RBC) membranes by using high-pressure liquid chromatography in the following groups: (1) 23 chronic renal failure (CRF) patients with creatine clearance (CCr)> 10 mL/min (26.2 +/- 16.7); (2) 21 end-stage CRF patients in conservative treatment with CCr <10 mL/min (6.8 +/- 1.8); (3) 30 hemodialysis (HD) patients; and (4) 30 healthy controls. RESULTS: The incorporation of CLA, CD18:3, and CD20:3 in RBC membranes was significantly reduced in group 1 and was even more reduced in groups 2 and 3. CLA significantly increased both in the plasma and adipose tissue of end-stage CRF patients only. CD18:3 and CD20:3 did not change in the plasma and adipose tissue of any group. No significant changes in linoleic acid and CD fatty acid hydroperoxides were found. CONCLUSIONS: The alterations of CD in CRF patients are not due to lipoperoxidation. The increased levels of CLA in plasma and adipose tissue of end-stage CRF patients may be due either to a reduced metabolization of CLA to CD18:3 and CD20:3, or to an altered site distribution with reduced incorporation in cellular membranes and accumulation in the plasma and adipose tissue. The clinical significance of these changes remains to be investigated.

Recognition and quantitation of cis-vaccenic and eicosenoic fatty acids in olive oils by 13C nuclear magnetic resonance spectroscopy.

The presence of 11-cis monoenoic fatty acids was detected in olive oil samples by means of 13C nuclear magnetic resonance spectroscopy, and the positional isomery on the glycerol backbone was derived. The 11-cis vaccenic and eicosenoic fatty acid resonances were recognized and the amounts of the fatty acids quantified. For comparison purposes, a quantitative analysis was also made by gas chromatography.

Decrease in linoleic acid metabolites as a potential mechanism in cancer risk reduction by conjugated linoleic acid.

Previous research suggested that conjugated linoleic acid (CLA) feeding during the period of pubescent mammary gland development in the rat resulted in diminished mammary epithelial branching which might account for the reduction in mammary cancer risk. Terminal end buds (TEB) are the primary sites for the chemical induction of mammary carcinomas in rodents. One of the objectives of the present study was to investigate the modulation of TEB density by increasing levels of dietary CLA and to determine how this might affect the risk of methylnitrosourea-induced mammary carcinogenesis. The data show a graded and parallel reduction in TEB density and mammary tumor yield produced by 0.5 and 1% CLA. No further decrease in either parameter was observed when CLA in the diet was raised to 1.5 or 2%. Thus, optimal CLA nutrition during pubescence could conceivably control the population of cancer-sensitive target sites in the mammary gland. Since both CLA and linoleic acid are likely to share the same enzyme system for chain desaturation and elongation, it is possible that increased CLA intake may interfere with the further metabolism of linoleic acid. Fatty acid analysis of total lipid showed that CLA and CLA metabolites continued to accumulate in mammary tissue in a dose-dependent manner over the range 0.5-2% CLA. There was no perturbation in tissue linoleic acid, however, linoleic acid metabolites (including 18:3, 20:3 and 20:4) were consistently depressed by up to 1% CLA. Of particular interest was the significant drop in 20:4 (arachidonic acid), which is the substrate for the cyclooxygenase and lipoxygenase pathways of eicosanoid biosynthesis. Thus the CLA dose-response effect on arachidonic acid suppression corresponded closely with the CLA dose-response effect on cancer protection in the mammary gland. This information is critical in providing new insights regarding the biochemical action of CLA.

An increase in vitamin A status by the feeding of conjugated linoleic acid.

Previous research indicated that conjugated linoleic acid (CLA) is a potent inhibitor of mammary carcinogenesis. The present study showed a progressive increase in retinol (vitamin A alcohol) in the liver in proportion to CLA intake in rats that were fed different levels of CLA (in increments of 0.5%) for 1 month. The escalation reached a magnitude of about fivefold over the control at 2% dietary CLA. In contrast, the increase in liver retinyl esters peaked at about twofold between 0.5% and 1% CLA. Only retinol was detected in mammary tissue; a maximal twofold increase was attained at 0.5% CLA, and no dose-response effect was evident. The above findings are discussed in relation to two important questions: 1) How does CLA raise vitamin A status in the animal? 2) Is the increase in vitamin A associated with the anticarcinogenic effect of CLA?

Inhibition of peroxynitrite dependent DNA base modification and tyrosine nitration by the extra virgin olive oil-derived antioxidant hydroxytyrosol.

Hydroxytyrosol is one of the o-diphenolic compounds in extra virgin olive oil and has been suggested to be a potent antioxidant. The superoxide radical (O2*-) and nitric oxide (NO*) can react very rapidly to form peroxynitrite (ONOO ), a reactive tissue damaging species thought to be involved in the pathology of several chronic diseases. Hydroxytyrosol was highly protective against the peroxynitrite-dependent nitration of tyrosine and DNA damage by peroxynitrite in vitro. Given that extra virgin olive oil is consumed daily by many humans, hydroxytyrosol derived from this diet could conceivably provide a defense against damage by oxidants in vivo. The biological activity of hydroxytyrosol in vivo will depend on its intake, uptake and access to cellular compartments.

Peroxidase-catalyzed oxidation of beta-carotene in HL-60 cells and in model systems: involvement of phenoxyl radicals.

Recent studies provide extensive evidence for the importance of carotenoids in protecting against oxidative stress associated with a number of diseases. In particular, reactions of carotenoids with phenoxyl radicals generated by peroxidase-catalyzed one-electron metabolism of phenolic compounds may represent an important antioxidant function of carotenoids. To further our understanding of the antioxidant mechanisms of carotenoids, we used in the present work two different phenolic compounds, phenol and a polar homologue of vitamin E (2,2,5,7,8-pentamethyl-6-hydroxychromane, PMC), as representatives of two different types of phenols to study reactions of their respective phenoxyl radicals with carotenoids in cells and in model systems. We found that phenoxyl radicals of PMC did not oxidize beta-carotene in either HL-60 cells or in model systems with horseradish peroxidase (HRP)/H2O2. In contrast, the phenoxyl radicals generated from phenol (by native myeloperoxidase in HL-60 cells or HRP/H2O2 in model systems) effectively oxidized beta-carotene and other carotenoids (canthaxanthin, lutein, lycopene). One-electron reduction of the phenoxyl radical by ascorbate (assayed by electron spin resonance-detectable formation of semidehydroascorbyl radicals) prevented HRP/H2O2-induced oxidation of beta-carotene. PMC, but not phenol, protected beta-carotene against oxidation induced by a lipid-soluble azo-initiator of peroxyl radicals. No adducts of peroxidase/phenol/H2O2-induced beta-carotene oxidation intermediates with phenol were detected by high-performance liquid chromatography-mass spectrometry analysis of the reaction mixture. Since carotenoids are essential constituents of the antioxidant defenses in cells and biological fluids, their depletion through the reaction with phenoxyl radicals formed from endogenous, nutritional and environmental phenolics, as well as phenolic drugs, may be an important factor in the development of oxidative stress.

Non-random peroxidation of different classes of membrane phospholipids in live cells detected by metabolically integrated cis-parinaric acid.

Quantitative assays of lipid peroxidation in intact, living cells are essential for evaluating oxidative damage from various sources and for testing the efficacy of antioxidant interventions. We report a novel method based on the use of cis-parinaric acid (PnA) as a reporter molecule for membrane lipid peroxidation in intact mammalian cells. Using four different cell lines (human leukemia HL-60, K562 and K/VP.5 cells, and Chinese hamster ovary (CHO) fibroblasts), we developed a technique to metabolically integrate PnA into all major classes of membrane phospholipids, i.e., phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and cardiolipin, that can be quantified by HPLC with fluorescence detection. Integrated PnA constituted less than 1% of lipid fatty acid residues, suggesting that membrane structure and characteristics were not significantly altered. Low concentrations (20-40 microM) of tert-butyl hydroperoxide (t-BuOOH) caused selective oxidation of PnA residues in phosphatidylserine and phosphatidylethanolamine of K562 cells and K/VP.5 cells while cell viability was unaffected. At higher t-BuOOH concentrations (exceeding 100 microM), however, a progressive, random oxidation of all major phospholipid classes occurred and was accompanied by significant cell death. In HL-60 cells, phosphatidylethanolamine, phosphatidylserine and cardiolipin were sensitive to low concentrations of t-BuOOH, while phosphatidylcholine and phosphatidylinositol were not affected. Phosphatidylinositol was the only phospholipid that responded to the low concentrations of t-BuOOH in CHO cells. At high t-BuOOH concentrations, again, all phospholipid classes underwent extensive oxidation. All phospholipids were nearly equally affected by peroxidation induced by a initiator of peroxyl radicals, 2,2'-azobis-(2,4-dimethylvaleronitrile) AMVN), in K562 cells. In gamma-irradiated (4-128 Gy) CHO cells, phosphatidylserine was the most affected phospholipid class (34% peroxidation) followed by phosphatidylinositol (24% peroxidation) while the other three phospholipid classes were apparently unaffected. Since loss of PnA fluorescence is a direct result of irreparable oxidative loss of its conjugated double bond system, the method described allows for selective and sensitive monitoring of oxidative stress in live cells without interference from cell repair mechanisms.

A novel approach to study linoleic acid autoxidation: importance of simultaneous detection of the substrate and its derivative oxidation products.

In this paper we have proposed a novel approach for studying the reaction of lipid oxidation by using the simplest chemical system available. Neat linoleic acid was incubated for 24 hours at 37 degrees C in the air. The course of lipid oxidation was followed by measuring simultaneously by HPLC with a diode array detector 1) linoleic acid decrease, 2) the products formed by radical attack, namely four hydroperoxy-octadeca-dienoic acid (HPODE) isomers, two c,t (c,t) and two trans,trans (t,t). 3) the byproducts formed by HPODE degradations, the four oxo-octadeca-dienoic acid (oxo-ODE) isomers. In HPODEs the presence of conjugated diene chromophore was confirmed by second derivative spectrophotometry. c,t HPODEs were also identified for their positional isomerism, while for t,t molecules the lack of suitable reference compound makes unfeasible the identification of their positional isomerism. As in the case of the latter two c,t and two t,t oxo-ODE isomers were characterized. This simple system appears to be useful for studying the activity exherted by lipophilic molecules that, like alpha-tocopherol, may act as antioxidants and/or as hydrogen atom donating molecules. The presence of alpha-tocopherol in different concentration for 24 hours in the reaction environment, shifts the reaction of linoleic acid autoxidation towards different byproduct formations. From the results obtained it is evident that alpha-tocopherol acts as hydrogen atom donor at all concentration tested, shifting the reaction toward a prevalent formation of c,t isomer of both HPODEs and oxo-ODEs. At concentration lower than 40 nmoles, when the ratio between alpha-tocopherol and linoleic acid was 1:100, the reaction of autoxidation is strongly inhibited, while at higher concentration alpha-tocopherol acted as a prooxidant. In these experimental conditions, alpha-tocopherylquinone was spectrophotometrically identified as the predominant oxidation product of alpha-tocopherol.

No direct evidence of increased lipid peroxidation in hemodialysis patients.

Lipid peroxidation, as measured by the thiobarbituric acid test, has been reported to have increased in hemodialysis (HD) patients, even though the test has low specificity in vivo. Conjugated diene fatty acid (CDFA) hydroperoxides are formed during lipid peroxidation, but not all conjugated dienes (CD) detected in humans originate from lipid peroxidation: octadeca-9,11-dienoic acid, a nonhydroperoxide CD derivative of linoleic acid (CDLA), has a dietary origin. We evaluated CDFA hydroperoxides, CDLA and linoleic acid, using high-performance liquid chromatography, in lipids extracted from plasma, adipose tissue and RBC membranes obtained from 25 patients treated with HD, 16 patients treated with hemodiafiltration (HDF) and 29 controls. No differences in the levels of CDFA hydroperoxides and linoleic acid were seen in any of the groups. Concentrations of CDLA were found to be significantly high in the adipose tissue and low in the RBC membranes of HD patients. HDF-treated patients showed the same results as HD patients. No direct evidence of increased lipid peroxidation was found in HD patients. This does not exclude the possibility that lipid peroxidation is increased and escapes direct detection due to the body's homeostatic control eliminating the increased production of hydroperoxides. Both HD- and HDF-treated patients showed a significant change in CDLA concentrations, either in the adipose tissue, or in the RBC membranes. These dietary CD may be mistaken for markers of lipid peroxidation by conventional methodologies.

Enhanced interfacial catalysis and hydrolytic specificity of phospholipase A2 toward peroxidized phosphatidylcholine vesicles.

The hydrolytic action of phospholipase A2 was examined with unilamellar vesicles composed of soybean phosphatidylcholine in terms of the calcium dependency of the enzyme and substrate specificity following lipid peroxidation. Experiments were performed under conditions where enzyme:substrate ratios were low, specifically in the range of one to five enzyme molecules for every 10 vesicle particles. Accordingly, low hydrolytic activities were found where less than 15% of the phospholipids were hydrolyzed under the various conditions of time, enzyme:substrate ratios, calcium concentrations, and extent of peroxidation utilized. Vesicle peroxidation increased the Ca2+ binding potential to a degree comparable to addition of the anionic phospholipid, dioleoylphosphatidic acid (DOPA). A remarkable similarity was found between the binding profiles for Ca2+ and phospholipase A2 activity; however, enzyme activity toward oxidized vesicles was beyond the increases observed for Ca2+ binding. Under conditions where approximately 5% of the phospholipids were peroxidized the effective Ca2+ concentration required for half-maximal activity was less than one-half that required for unoxidized vesicles. Peroxidation of vesicle phospholipids markedly increased the rate and extent of hydrolysis, even in the presence of DOPA or deoxycholate. Deoxycholate is known to induce vesicle fusion such that a larger proportion of enzyme is associated with a fewer number of enlarged vesicles. Using a dual isotope technique to measure hydrolysis of oxidized vs unoxidized phospholipids and covesicle preparations to study enzyme binding and activity, a significantly greater apparent intervesicle exchange of enzyme was found after peroxidation of vesicles with more than a twofold hydrolytic specificity toward the oxidized phospholipids. We postulate that a combination of structural and Ca2+ binding affinity changes are produced in membranes following lipid peroxidation which evoke an additive effect on PLA2 activity. Although oxidized phospholipids may serve as activators of phospholipase A2 by presenting the interface in a form where Ca2+ and enzyme binding and/or specific activity are increased, an additional and important factor appears to involve membrane fusion or vesicle-vesicle interactions. This process facilitates enzyme activity through the replenishment of substrates wherein the otherwise limited interaction of enzyme and substrate is overcome by more rapid or extensive vesicle fusion which increases access to the phospholipids available in the preparation.

Peroxidation and phospholipase A2 hydrolytic susceptibility of liposomes consisting of mixed species of phosphatidylcholine and phosphatidylethanolamine.

The relationship between lipid peroxidation and phospholipase A2 (PLA2) hydrolytic activity was studied using unilamellar vesicles (liposomes) as model membranes. Hydrolytic specificity was examined using vesicles prepared with pure bovine heart phosphatidylcholine (PC), bovine heart phosphatidylethanolamine (PE), or mixtures of these phospholipids, using two preparative procedures, i.e., sonication or extrusion. Lipid peroxidation was induced by incubating vesicles with cumene hydroperoxide and hematin at 37 degrees C. Determinations of the extent of peroxidation by means of diene conjugate content derived from second derivative spectra or by polarographic measurement of oxygen consumption rates provided a basis for comparing the extent of peroxidation of each phospholipid species to their subsequent hydrolysis by PLA2 (from Crotalus adamanteus). The extent of hydrolysis was determined through the release of arachidonic acid from either PC or PE. The PE distribution among the outer vs. inner leaflet of the membrane bilayer was nearly equal in sonicated vesicles, whereas most of the phospholipid was incorporated into the inner leaflet in extruded vesicles. The proportion of PE found in the inner leaflet progressively increased as the ratio of PE to PC increased in both sonicated and extruded vesicle preparations. Lipid peroxidation had no effect on PE distribution under the conditions examined. There was a clear preference for PC peroxidation for all vesicle compositions tested and PC was preferentially hydrolyzed by PLA2. This effect is proposed to result from a perturbation of membrane structure following peroxidation with assimilation of PC into PLA2-susceptible domains whereas PE peroxidation and hydrolysis is less affected in mixed PC/PE vesicles. Lipid peroxidation imposes an additional hydrolytic susceptibility over the effects exerted through the mixing of these phospholipids which is based on structural changes rather than formation of specific substrates for PLA2.

On the hepatotoxicity of 1,1,2,2-tetrachloroethane.

Intoxication of male and female mice with a single dose (300 or 600 mg/kg) of 1,1,2,2-tetrachloroethane (TTCE) resulted in significant decreases in cytochrome P-450 (to 58-73% of the control) and NADPH-cytochrome (P-450) c-reductase (to 29-35% of the control) in hepatic microsomes. This was accompanied by an alteration of mixed function monooxygenases stemming from the marked reduction (to 20-64% of the control) of several oxidative activities to selected substrates towards different P-450 isozymes (classes IA1, IA2, IIB1, IIE1 and IIIA). As phase II markers, epoxide hydrolase (approximately 35% loss), UDP-glucuronosyl transferase (approximately 42% loss) and to a lesser extent glutathione S-transferase (approximately 17% loss) were all affected. Also, the activity of delta-aminolevulinic (ALA) synthetase was decreased (approximately 57% of the control). On the contrary, heme oxygenase activity was increased (up to 35%) at the maximal dose tested. The decrease of P-450-function may be explained in terms of an alteration in the rate of heme biosynthesis and degradation, provoking a loss of heme content (approximately 33%) as well as of the direct inactivation of both P-450 and reductase. Because of increasing evidence on the involvement of free radical intermediates in the case of toxicity of haloalkanes, electron spin resonance spectroscopy (ESR) spin-trapping in vivo techniques were used to characterize the possible free radical species involved in the observed liver damage. The results obtained with the spin-trap N-benzylidene-2-methylpropylamine N-oxide (phenyl t-butylnitrone, PBN) provide evidence for the formation and trapping of the CHCl2CHCl free radicals. The detection of conjugated diene signals by means of second-derivative spectrophotometry, have enabled us to show that in vivo lipid peroxidation may be one of the main mechanisms responsible for TTCE hepatotoxicity.

In vivo and in vitro evidence concerning the role of lipid peroxidation in the mechanism of hepatocyte death due to carbon tetrachloride.

Isolated rat hepatocytes exposed to CCl4 showed a stimulated formation of malonaldehyde after only 30-60 min incubation. Conversely, the onset of hepatocyte death was a relatively late event, being significant only after 2-3 h of treatment. A cause-effect relationship between the two phenomena has been demonstrated by using hepatocytes isolated from rats pretreated with alpha-tocopherol. Comparable results were obtained in vivo where supplementation with alpha-tocopherol 15 h before CCl4 dosing induced a partial or complete protection against the drug's necrogenic effect, depending on the concentration of the haloalkane used. Moreover, the vitamin supplementation prevented the CCl4-induced increase of liver total calcium content, probably by blocking alterations in the liver cell plasma membranes due to lipid peroxidation.

Serum levels of tumor-associated trypsin inhibitor (TATI) in benign and malignant gynecological diseases.

The behavior of tumor-associated trypsin inhibitor (TATI) as a marker for gynecological cancer was studied in a control population and in patients with different benign and malignant diseases. When a cut-off level of 21.4 micrograms/l was used the specificity was 100% in patients with benign diseases. The sensitivity in patients with malignant tumors was low for cervical and corpus cancer, 13% and 14%, respectively, whereas it was 33% in all the ovarian malignant tumors, reaching 60% in the mucinous type. There was a clear correlation between TATI level and stage.

Conjugated diene and trans fatty acids in a choline-devoid diet hepatocarcinogenic in the rat.

It has been postulated that the hepatocarcinogenicity of a choline-devoid diet in rats stems from peroxidation of liver lipids. We have investigated whether the diet contains conjugated dienes that could account directly for those detected in liver lipids of rats fed a choline-devoid diet. Analyses were performed on samples of corn oil and of a partially hydrogenated fat used to prepare semipurified choline-devoid and choline-supplemented diets, and on fat extracted from two pairs of diets, one set containing 5% corn oil and 10% partially hydrogenated fat, and the other only corn oil (15%). The analyses consisted of quantitation of conjugated dienes by UV spectrophotometry, separation of fatty acids with conjugated dienes by HPLC, and quantitation of trans fatty acids by IR spectrophotometry. Small levels of conjugated diene and trans fatty acids were present in the corn oil, but much higher amounts were found in the partially hydrogenated fat. HPLC analysis yielded distinct elution profiles for the fatty acids with conjugated dienes present in the two fats, and similar results were obtained with fat extracted from the diets. However, no differences were observed between choline-devoid and control choline-supplemented diets. The results indicate that caution must be exercised in interpreting data from UV analysis of tissue lipids of rats fed diets containing a partially hydrogenated fat.

Conjugated diene and trans fatty acids in tissue lipids of rats fed an hepatocarcinogenic choline-devoid diet.

Groups of male Fischer-344 rats were fed two pairs of semi-purified choline-devoid and choline-supplemented diets, one set containing 5% corn oil and 10% partially hydrogenated fat, and the other only corn oil (15%). Analyses were performed on lipids extracted from whole liver, liver nuclei and adipose tissue of the rats. The analyses consisted of quantitation of conjugated dienes by UV spectrophotometry, separation of fatty acids with conjugated dienes by HPLC and quantitation of trans fatty acids by IR spectrophotometry. Conjugated dienes and conjugated diene and trans fatty acids were observed in adipose tissue total lipids, at concentrations that reflected those in the diets fed. The same was true of trans fatty acids in liver lipids. However, no conjugated dienes, or fatty acids with conjugated dienes, were detected in liver lipids of rats fed the diets formulated with only corn oil. In contrast, conjugated dienes were detected in total and neutral lipids, but not in phospholipids, of whole liver and liver nuclei of rats fed the diets formulated with partially hydrogenated fat. The neutral lipids contained fatty acids with conjugated dienes that eluted with the retention time of conjugated diene fatty acids, present in the dietary partially hydrogenated fat. It is concluded that a choline-devoid diet, which is hepatocarcinogenic in the rat, does not induce a peroxidation of liver cell membrane lipids, and that not only trans fatty acids, but also fatty acids with conjugated dienes present in a partially hydrogenated fat, are absorbed and assimilated in rat tissue lipids.