Lipid components and water soluble metabolites in salted and dried tuna (Thunnus thynnus L.) roes.

The salted and dried product of tuna roe (bottarga) is a seafood characteristic of the Mediterranean area and exported all over the world. Samples of bottarga from bluefin tunas (Thunnus thynnus, L.) caught in the southwest Mediterranean sea were analysed. The samples were characterised by high content of marine wax esters (55-67 mol% of lipid classes), of docosahexaenoic (22:6 n-3, 25 w%) and oleic (18:1 n-9, 19 w%) fatty acids. Cholesterol was detected as 7-9 w% of lipids. Free fatty acids, index of lipid hydrolysis, represented 32-39 mol% over total fatty acids. Among metabolites, nutrients as taurine, nicotinamide and ß-alanine, were found. The microflora comprised staphylococci, enterococci (2.2 log(10)CFU/g) and lactic acid bacteria (3 log(10) CFU/g). The food-borne pathogens Staphylococcus aureus, Listeria monocytogenes and Salmonella spp. were not detected. These findings indicate tuna bottarga as valuable source of nutrients.

Extra virgin olive oil phenolics: absorption, metabolism, and biological activities in the GI tract.

Olive oil, a typical ingredient of the Mediterranean diet, possesses many beneficial health effects. The biological activities ascribed to olive oil consumption are associated in part to its phenolics constituents, and mainly linked to the direct or indirect antioxidant activity of olive oil phenolics and their metabolites, which are exerted more efficiently in the gastrointestinal (GI) tract, where dietary phenolics are more concentrated when compared to other organs. In this regard, we present a brief overview of the metabolism, biological activities, and anticancer properties of olive oil phenolics in the GI tract.

Protective effect of hydroxytyrosol and tyrosol against oxidative stress in kidney cells.

Bioavailability studies in animals and humans fed with extravirgin olive oil demonstrated that hydroxytyrosol and tyrosol, the major simple phenolic compounds in extravirgin olive oil, are dose-dependently absorbed and excreted. Once absorbed, they undergo extensive metabolism; hydroxytyrosol and tyrosol concentrate mainly in the kidney, where they may exert an important role in the prevention of oxidative stress induced renal dysfunction. In this study we monitored the ability of hydroxytyrosol and tyrosol to protect renal cells (LLC-PK1) following oxidative damage induced by H2O2. Oxidative stress was evaluated by monitoring the changes of the membrane lipid fraction. Hydroxytyrosol exerted a significant antioxidant action, inhibiting the production of MDA, fatty acids hydroperoxides and 7-ketocholesterol, major oxidation products of unsaturated fatty acids and cholesterol, and thus protecting the cells from H2O2-induced damage. Tyrosol, instead, in this experimental model, did not exert any protective effect.

13C NMR, GC and HPLC characterization of lipid components of the salted and dried mullet (Mugil cephalus) roe "bottarga".

13C NMR spectroscopy, in conjunction with HPLC and GC techniques, has been used to study the molecular composition of lipids extracted from commercial products of bottarga. To this goal, both the saponifiable and unsaponifiable fractions of lipid extracts were also examined by 13C NMR. Among the major lipid classes wax esters (WE) showed a concentration of more than 50mol%, triacylglycerols (TAG) and phospholipids (PL) represented a minor fraction. Concentrations up to 29mol% of free fatty acids (FFA) were found. The most represented fatty alcohol was 16:0 that accounted for more than 50%, among fatty acids the most represented were 16:1 n-7, 22:6 n-3, 18:1 n-9, 16:0, and 20:5 n-3, in particular the n-3 polyunsaturated fatty acids (PUFA) averaged 40mg/g of the edible portion. 13C NMR spectroscopy put in evidence that cholesterol was present in its free and esterified forms and its total content was measured as ca. 10mg/g of the edible portion.

The effect of ferric-nitrilotriacetic acid on the profile of polyunsaturated fatty acids in the kidney and liver of rats.

Intraperitoneal injection of the iron-complex, ferric-nitrilotriacetate (Fe-NTA), induces renal proximal tubular damage associated with oxidative damage in vivo. Fe-NTA induced a time-dependent decrease of several polyunsaturated fatty acids (PUFA), together with increased conjugated diene values and decreased cellular levels of alpha-tocopherol and glutathione. At the time of maximum detectable oxidation (3 h), after the injection of a sublethal dose of Fe-NTA there were clear reductions in the peak values over the controls for several fatty acids notably, 20:5 (eicosapentaenoic acid) (36%), 22:6 (docosahexanoic acid) (30%), 20:3 n6 (eicosatrienoic acid) (30%) and 20:4 (arachidonic acid) (28%) in the kidney. Fewer fatty acids showed a reduction in their residual values in the liver. 20:5 was reduced by 45% and for the 18:3 n3 and 18:3 n6, reductions of 35%, respectively. The profile of PUFAs is sensitive to the oxidative damage due to Fe-NTA and this may find applications as oxidative biomarker model.

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.

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.

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.

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.

Analysis of sodium-23 nuclear magnetic resonance spin-lattice relaxation for the study of the intracellular sodium state.

Sodium-23 Nuclear Magnetic Resonance relaxation spectroscopy has been used to investigate the state of intracellular Na+ in control and CCl4-treated rat livers. The analysis of spin-lattice relaxation rates at 1.88 and 7.07 Tesla based on a two-site exchange model led to estimates of pertinent modulation times. Also it has been found that a relatively high quantity of Na+ (PB = 1.59 x 10(-2)) is bound to charged sites of intracellular macromolecules or membranes. The degree of binding strongly decreases in CCl4 treated rat livers.

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.

Free radicals and lipid peroxidation in liver of rats kept on a diet devoid of choline.

Rodents kept on a choline devoid (CD) diet up to 14 months develop hepatic lesions progressing through two broad stages. The first is characterized by severe steatosis and increase in cell turnover, the second by a gradual clearance of the deposited fat and fibrosis. Hepatocellular carcinomas eventually arise in rats fed for over 12 months, even though the animals aer not exposed to chemical carcinogens. It has been suggested that the diet may trigger generated thereby may be responsible for initiation of liver cancer and promotion. The radicals would lead to DNA damage, and the altered DNA in a proliferating liver would result in initiation of the carcinogenic process. In this communication we present evidence that the diet used in the above studies contained stable fatty acid isomers with conjugated dienes, which are absorbed and deposited in rat liver. This finding cast doubts on whether a CD diet does indeed cause a peroxidation of cellular membrane lipids. Electron spin resonance (ESR) spectroscopy was also used to investigate whether any abnormal pattern of free radicals exists in the liver of rats fed a CD diet. No significant differences were noted in ESR spectra of either transition metal-centered signals, or organic free radicals.

Conjugated dienes detected in tissue lipid extracts by second derivative spectrophotometry.

By studying lipid peroxidation induced by tetrachloromethane in rat liver microsomal PUFA, it has recently been shown that the primary products formed are conjugated diene hydroperoxides having either cis, trans (c,t) or trans,trans (t,t) stereochemistry. Both c,t and t,t hydroperoxidienes present distinct absorbances at 242 nm and 233 nm, respectively. The reaction is kinetically controlled in relation to the total H-atom donating ability of the cell environment. These results have been confirmed in vivo and in vitro experiments performed under different experimental conditions. The need for a precise and objective method to detect conjugated diene signals, the inherent difficulties with current techniques, and the availability of new spectrophotometric techniques have led us to devise a new method based on the second derivatization of the spectrum.

Free-radical metabolism of carbon tetrachloride in rat liver mitochondria. A study of the mechanism of activation.

Alterations in liver mitochondria as consequence of rat poisoning with carbon tetrachloride (CCl4) have been reported over many years, but the mechanisms responsible for causing such damage are still largely unknown. Isolated rat liver mitochondria incubated under hypoxic conditions with succinate and ADP were found able to activate CCl4 to a free-radical species identified as trichloromethyl free radical (CCl3) by e.s.r. spectroscopy coupled with the spin-trapping technique. The incubation of mitochondria in air decreased free-radical production, indicating that a reductive reaction was involved in the activation of CCl4. However, in contrast with liver microsomes (microsomal fractions), mitochondria did not require the presence of NADPH, and the process was not significantly influenced by inhibitors of cytochrome P-450. The addition of inhibitors of the respiratory chain such as antimycin A and KCN decreased free-radical formation by only 30%, whereas rotenone displayed a greater effect (approx. 84% inhibition), but only when preincubated for 15 min with mitochondria not supplemented with succinate. These findings suggest that the mitochondrial electron-transport chain is responsible for the activation of CCl4. A conjugated-diene band was observed in the lipids extracted from mitochondria incubated with CCl4 under anaerobic conditions, indicating that stimulation of lipid peroxidation was occurring as a result of the formation of free-radical species.

MPTP fails to induce lipid peroxidation in vivo.

It has been speculated that the conversion of MPTP to MPP+ destroys dopaminergic neurons by promoting the generation of hydroxyl radicals and causes lipid peroxidation. The results obtained in the present work indicate that the primary products of lipid peroxidation are not detectable in MPTP treated animals and thus other mechanisms besides lipid peroxidation should be considered to explain the cytotoxicity of this neurotoxin.

NMR spin-lattice relaxation times of intracellular Na-23 on rat livers and related lipid peroxidation following CCl4 intoxication.

Liver tissues were isolated from rats acutely intoxicated with carbon tetrachloride, and Na-23 NMR signals were analyzed to investigate the T1 relaxation times of intracellular sodium ions under pathological conditions in presence of the paramagnetic shift reagent (dysprosium tripolyphosphate). We studied the significant increase of T1 found in CCl4 treated rats with respect to controls, which was elsewhere demonstrated as being independent of cell necrosis. Evidence is given that neither fat accumulation nor proliferative processes affect the observed T1 lengthening. When T1 relaxation times were measured in the liver of vitamin E treated rats subsequently intoxicated with carbon tetrachloride, a significative shortening of T1 with respect to CCl4-intoxicated rats was observed. These results were discussed in terms of the antioxidant action exerted by vitamin E, taking into account that peroxidation of microsomal lipids is the key factor in the process of carbon tetrachloride induced liver injury. Furthermore, the observed T1 changes were discussed in terms of the interactions of Na+ with cell membranes and/or the occurrence of viscosity changes.

Na-23 NMR spin-lattice relaxation times in rat tissues, and related modifications following CCl4 intoxication.

Intracellular Na-23 NMR signals were analyzed to investigate the T1 nuclear magnetic relaxation times of sodium ions in rat tissues under physiological conditions in presence of the shift reagent Dy(PPPi)2(7-). The T1 values so measured showed a high degree of reproducibility (10%). The order of T1 values found for the investigated rat tissues was the following: liver, spleen less than kidney less than muscle, brain less than blood. Furthermore, livers of rats treated with the known hepatotoxic agent CCl4 were studied. This tissue showed a significative increase of T1 with respect to those of normal rats. The T1 lengthenings were discussed in terms of various cellular injuries induced in the liver by CCl4 intoxication and our results showed that T1 lengthening did not depend on liver necrosis.

[Further investigations on the protective effect of cadmium acetate in bromotrichloromethane poisoning]. / Ulteriori indagini sul'effetto protettivo dell'acetato di cadmio nella intossicazione da triclorobromometano.

The rats were pretreated with cadmium acetate (20 mg/Kg.ip) and subsequently challenged with a dose of bromotrichloromethane (2,6 mmoles/Kg.po), the haloalkane produced no change on liver polyribosomes, as evidenced by disaggregation of sedimentation profile. Cadmium acetate failed to protect against CBrCl3-induced lipid peroxidation, as measured by malonaldehyde production of liver homogenate.