Assessment of in vitro removal of cholesterol oxidation products by Lactobacillus casei ATCC334.

Cholesterol oxidation products (COPs) are a group of compounds formed during processing and storage of foods from animal origin. After ingestion, COPs are absorbed in the intestine and can be distributed to serum and various tissues, potentially promoting a variety of toxic effects. Therefore, inhibition of their intestinal absorption may contribute to reduce the health risks associated with dietary intake of COPs. Some studies have shown that drugs and dietary compounds may inhibit the intestinal absorption of dietary COPs. However, proven cholesterol- and/or food toxins-binding lactic acid bacteria have not been previously evaluated as potential COPs removal agents. The aim of this study was to assess the ability of Lactobacillus casei ATCC334 to remove COPs in aqueous solution. Results showed the ability of both growing and resting cells to remove COPs (ca. 30-60%). All COPs-bacterium interactions were specific and partly reversible, being resting cells the most efficient for COPs removal in a ranking order of 7-KC > 7α-OH/7ß-OH > triol > 5,6ß-EP > 5,6α-EP > 25-OH. Binding to the cell wall and/or cell membrane incorporation appears to be the most likely mechanisms involved on COPs removal by L. casei ATCC 334.

In vivo assessment about the effects of a diet containing iodine-enriched foodstuffs. A pilot study in 30 volunteers.

BACKGROUND: Iodine deficiency (ID) still now represents one of the major worldwide health problems. ID is the result of insufficient dietary iodine intake. Iodine is an essential micronutrient but scarcely present in nature. The main strategy for the correction of ID is the fortification of table salt with iodide/iodine but Italy is far from reaching an iodized salt use higher 90% of population. Also because of the evidence for the risk on blood pressure, it is recommended to decrease the daily salt intake to less than 5 g/d. An opportunity to increase the iodine intake is the possibility to introduce iodine fortification in the industrial processing of foods. AIM: The aim was to evaluate the effectiveness of a diet containing iodized foods enriched during industry processing with protected iodized salt (Presal®). SUBJECTS AND METHODS: The evaluation of increasing of iodine intake was assessed by measuring the urinary iodine excretion (UIE) in 30 healthy volunteers who added to their alimentary habits a basket of iodine-enriched foodstuffs. RESULTS: Median UIE at baseline was 105 µg/l, 156 µg/l during the enriched diet and 90.5 µg/l a week after withdrawal of enriched diet. CONCLUSIONS: Stable iodized salt (Presal®) represents a good way to introduce iodine with the normal diet without increasing the normal consumption of salt for the healthy problems related to the blood pressure. The availability of stable iodized salt (Presal®) allows the preservation of iodine after cooking.

[Milk and butter. From the Neolithic to the current nutritional aspects]. / Il latte e il burro. Dal Neolitico agli attuali aspetti nutrizionali.

The evolution in the history of nutrition knowledge towards dairy products, is strictly related to the socio-cultural development of humans. In fact, milk and butter have accompanied humans since ancient times, which traces of the consumption of such products are dated back about the earliest times after the last (glaciation) ice age, while the application for extra nutritional uses, such as cosmetics and ceremonial rites, are reported in the writings of the Old Testament. Even in Italy, before the Roman Empire, were known rudimentary techniques of production and storage of dairy products. But only with the advent of the Etruscans, and the Romans later, that the use of milk and dairy products reach a wide diffusion in several applications. Since the advent of Christ until today, milk and its derivatives have maintained a privileged place in the human diet, but it is only with the advent of modern medicine and new findings in lipidic chemistry that emerged multiple biological and nutritional properties, very important for human health. After a short summary of the ancient history of the milk and butter, the role of dairy products in cancer, in hypercholesterolemia, and cardiovascular disease are reported. Moreover, the current opinions on saturated fatty acids, the role of polyunsaturated fatty acids and their lipid mediators obtained by the action of cyclooxygenase, lipoxygenase and the cytochrome P450 enzymes, are treated. Even if sometimes mistreated, the milk, but most of all its high fat content derivatives such as butter, is a rich source of biologically active compounds that foster a controversial action against neolplastic and cardiovascular disease. These compounds, mainly contained in the lipid fraction, for the more obvious relationships that exist between nutrition and health status, have been the subject in the last decades of intense scientific investigation in which there were expressed lights and shadows, but recognizing that not all fats are harmful and further thorough studies are necessary, in particular, on the derived lipid mediators. This will allow a significant progress based on new scientific evidences, further orienting researchers and clinicians on evidence-based nutritional science.

Evaluation of the oxidative status of virgin olive oils with different phenolic content by direct infusion atmospheric pressure chemical ionization mass spectrometry.

Atmospheric pressure chemical ionization mass spectrometry was used to predict the oxidative status of virgin olive oils (VOO) during their storage. VOO samples, with and without phenolic compounds, were stored in the dark at 60 degrees C up to 7 weeks. The VOO samples were diluted in an alkaline propanol/methanol mixture and directly infused into an ion-trap mass spectrometer. The abundances of the [M-H](-) peaks of free fatty acids, oxidized fatty acids, tocopherols and phenolic compounds, jointly with their oxidized forms, were measured and used as predictors. Two linear discriminant analysis (LDA) models were constructed in order to classify samples according to their oxidative levels. The first model was constructed using both VOO samples (with and without phenols), considering as predictors only fatty acids and their oxidized products. The second LDA model was constructed with the VOO sample with phenolic compounds considering as predictors all the peaks measured. In both models, the samples divided in the eight different storage times were correctly classified (100%) by leave-one-out cross-validation with an excellent resolution among all the category pairs (for the first model Wilks' lambda, lambda(w) = 0.229 and for the second lambda(w) = 0.928). This method is a very fast tool for on-line monitoring of VOO oxidation status.

Platelet Stearic Acid in different population groups: biochemical and functional hypothesis

As part of a complex work about the characterization of Coronary Heart Disease by platelet fatty acids using a Self Organizing Map (Artificial Neural Network), particular aspects of the stearic acid platelet concentration have been found. The Authors look at the biochemical and functional aspects of Stearic on platelets, and, even in consideration of a considerable discrepancy in the levels of stearic acid in some groups of the subjects investigated, attempt a plausible interpretation. It is stressed the evidence, however, that the stearic acid is high in patients with ischemic heart disease.This, in the opinion of the authors, doesn’t necessary means that stearic acid is a negative finding in Coronary heart Disease (AU)

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Evaluation of the antioxidant capacity of individual phenolic compounds in virgin olive oil.

Virgin olive oil has a high resistance to oxidative deterioration due to its tryacylglycerol composition low in polyunsaturated fatty acids and due to the presence of a group of phenolic antioxidants composed mainly of polyphenols and tocopherols. We isolated several phenolic compounds of extra virgin olive oil (phenyl-ethyl alcohols, lignans, and secoiridoids) by semipreparative high-performance liquid chromatography (HPLC) and identified them using ultraviolet, atmospheric pressure chemical ionization, and electrospray ionization MS detection. The purity of these extracts was confirmed by analytical HPLC using two different gradients. Finally, the antioxidant capacity of the isolated compounds was evaluated by measuring the radical scavenging effect on 1,1-diphenyl-2-picrylhydrazyl radical, by accelerated oxidation in a lipid model system (OSI, oxidative stability instrument), and by an electrochemical method.

Magnetic resonance spectroscopy and chromatographic methods identify altered lipid composition in human renal neoplasms.

We report on the characterization of the lipid obtained from cortical and medullary normal human kidney tissue, benign renal neoplasms (oncocytoma) and 2 different types of malignant renal neoplasms (chromophobic cell carcinoma and clear cell carcinoma). The total lipid fractions were analyzed by 13C magnetic resonance spectroscopy and thin-layer chromatography, whereas the composition of the total fatty acids and the content of total cholesterol were determined by gas chromatography. alpha-Tocopherol was detected and quantified by high-performance liquid chromatography. The spectroscopic and chromatographic analysis revealed significant differences in the renal tissues examined. It was confirmed that cholesteryl esters (mainly oleate) are typical of clear cell renal carcinomas. Their potential role as prognostic and diagnostic factors is discussed, with particular emphasis on its capability to indicate the tumor diffusion in healthy renal parenchyma. alpha-Tocopherol is prevalent in clear cell carcinoma and it is present in nearly the same low amounts in cortex, medulla and chromophobic cell renal carcinoma. Q10 coenzyme and dolichols were detected by thin-layer chromatography and they are present in significant amounts in the cortex and the benign oncocytoma. Great variations were found in the distribution of saturated and unsaturated fatty acids, especially in the docosapentaenoic, docosahexaenoic and arachidonic acids and the corresponding omega-6/omega-3 fatty acids ratio.

Calculation of the molar absorptivity of polyphenols by using liquid chromatography with diode array detection: the case of carnosic acid.

Antioxidant activity of vegetable extracts is related to the nature and the amount of active components, mainly polyphenols; therefore, a correct quantification of these molecules should be required to define their concentration in such kind of vegetable extracts. A fast and accurate method to calculate molar absorption coefficients (epsilon), by using HPLC, has been tested on standard polyphenols and caffeine, and should be widely adapted for standardless quantitative analysis. Molar absorptivity (epsilon) of carnosic acid (CA) was determined from 200 to 300 nm, by the proposed method and those values were compared to tert-butyl-hydroxytoluene (BHT) ones for further comparative quantification.

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.

Pressurized liquid extraction of lipids for the determination of oxysterols in egg-containing food.

Pressurized liquid extraction (PLE, ASE) was compared with the Folch procedure (a solid-liquid extraction with chloroform/methanol 2:1, v/v) for the lipid extraction of egg-containing food; the accuracy of PLE for the quantitative determination of oxysterols in whole egg powder was evaluated. Samples of spray-dried whole egg, an Italian vanilla cake (Pandoro) and egg noodles were used. Two different extraction solvents (chloroform/methanol 2:1, v/v, and hexane/isopropanol 3:2, v/v) were tested at different extraction temperatures and pressures (60 degrees C at 15 MPa, 100 degrees C at 15 MPa, 120 degrees C at 20 MPa). No significant differences in the lipid recovery of the egg powder sample using PLE were found. However, PLE of the vanilla cake and egg noodles with the chloroform/methanol mixture was not selective enough and led to the extraction of a non-lipid fraction, including nitrogen-containing compounds. In the same samples, the pressurized hexane/isopropanol mixture gave a better recovery result, comparable to that obtained using the Folch method. Cholesterol oxidation products of the Folch extract and the pressurized liquid extract of spray dried egg powder (obtained with hexane/isopropanol 3:2, v/v, at 60 degrees C and 15 MPa) were determined by gas chromatography. PLE performed under these conditions is suitable to replace the Folch extraction, because the differences between the two methods tested were not statistically significant. Moreover, PLE shows important advantages, since the analysis time was shortened by a factor of 10, the solvent costs were reduced by 80% and the use of chlorinated solvents was avoided.

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.

Mass spectral fragmentations of cholesterol acetate oxidation products.

In this work, electron-impact mass spectroscopy (EI-MS) was employed on a wide range of sterol compounds in order to study their behaviour with regard to their functional groups. In particular, some specific mechanisms of fragmentation occurring in these substrates (i.e. retro-Diels-Alder reaction, neutral molecules eliminations, specific hydrogen migrations) were investigated. Loss of the alkyl side chain and of the D ring were observed in all cases. Finally, a classification of sterols on the basis of characteristic mass spectral fragments is suggested, and further applications to substrates with functional groups on positions other than the A and B rings is proposed.

Chromatographic analysis of unsaponifiable compounds of olive oils and fat-containing foods.

The analysis of the "minor components" present in food lipids is usually hampered by the large diversity of compounds found in this fraction. High-purity degree reagents and solvents, good collection techniques and highly sensitive analysis are required in order to accurately identify and quantify these components. Chromatographic techniques have proven to be particularly suitable for these determinations, especially capillary gas chromatography. This study reports several analytical cases of the main classes of components of the unsaponifiable matter obtained from olive oils or food matrices.

Determination of furan fatty acids in extra virgin olive oil.

The presence of 4 different furan fatty acids (F-acids) was detected in 18 samples of transmethylated monovarietal extra virgin olive oil: methyl 10,13-epoxy-11,12-dimethyloctadeca-10,12-dienoate [diMeF(9,5)], methyl 12,15-epoxy-13,14-dimethyleicosa-12,14-dienoate [diMeF(11,5)] and both olefinic derivatives of diMeF(11,5) with one unsaturation on the side chains conjugated with the furan ring. Transmethylated oils were analyzed by normal phase high-performance liquid chromatography coupled on-line with capillary gas chromatography. After the gas chromatographic separation step, a more selective detection of F-acids was achieved by using a photoionization detector mounted in series with a flame ionization detector. The concentration of F-acids ranged between 50 ppb (detection limit of the method) and 2.1 ppm in the oil. The olefinic derivatives of diMeF(11,5) acids detected were not artifacts created during the sample preparation or during the chromatographic analysis.

Effect of different cooking methods on some lipid and protein components of hamburgers.

The effects of different cooking methods on the lipid and protein fractions of hamburger were evaluated. The lipid component was subjected to the following analyses: peroxide value; p-anisidine; total and free fatty acids; cholesterol and its oxidation products (quantified as 7-ketocholesterol). Lysinoalanine (LAL), free amino acids and D-amino acids (D-AA) were also determined in the protein fraction. All results were compared with a raw control. No significant differences were found among the cooking treatments with respect to D-AA and LAL. The degree of proteolysis, lipolysis and lipid oxidation varied depending on the treatment conditions. Regarding cholesterol oxidation, the combination of roasting and microwave heating caused more oxidation than the other treatments. The raw meat, however, showed an advanced degree of oxidation (25.2 ppm of total 7-ketocholesterol/120 g ground meat).

Determination of polycyclic aromatic hydrocarbons in water and water-based alcoholic beverages.

This paper proposes a simple HPLC method for the determination of polycyclic aromatic hydrocarbons (PAHs) in water, wine and beer. Samples were purified by PAH collection in solid-phase extraction (SPE) and analysed by reversed-phase HPLC (Supelcosil LC-PAH column from Supelco). For the beer sample, recoveries amounted to 28% for naphthalene and varied from 57% to 103% for the other PAHs; results are quantitative starting from fluoranthene (FI, the seventh component eluted). Almost all the beer and wine samples showed the presence of benzo(b)fluoranthene (BbF), benzo(k)fluoranthene (BkF), benzo(a)pyrene (BaP), benz(ghi)perylene (BghiP) and indeno(1,2,3-cd)pyrene (IP), and in some cases there were traces of FI, benzo(a)anthracene (BaA) and dibenz(ah) anthracene (DBahA). Total contents of PAHs ranged from trace amounts to 0.72 ppb. Traces of BbF, BkF, BaP, BghiP and IP were also found in the wine samples.

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.

High-performance liquid chromatography separation and light-scattering detection of phospholipids from cooked beef.

A sensitive high-performance liquid chromatography (HPLC) method for the separation and quantitative analysis of major phospholipids (PLs) in biological systems is described. PLs were purified by solid-phase extraction with an amino (NH2) phase. Separation of PLs was carried out on an HPLC silica gel column, with a mobile phase consisting of chloroform, methanol and ammonium hydroxide, and detection was performed with a light-scattering evaporative detector. HPLC analysis of PLs extracted from ground beef cooked under different conditions and capillary gas chromatography of the fatty acid methyl esters showed that cooking treatments did not have a significant effect on the PL composition and fatty acid contents of the single PLs in ground beef.

Identification of thermal oxidation products of cholesteryl acetate.

The polar products separated by solid-phase extraction from the peroxidation mixture of cholesteryl acetate, were investigated. The oxidation products were identified by comparing GC retention times as well as the mass spectra against those of available or synthesized standards. The main oxidation products where 7 beta-chydroperoxicholesteryl acetate, 7 alpha-hydroperoxicholesteryl acetate, 7-ketocholesteryl acetate, the alpha and beta isomers of 7-hydroxycholesteryl acetate, the alpha- and beta-epoxy isomers in 5,6 position and several derivatives from the loss of groups (especially the acetic and/or hydroxyl groups in the form of acetic acid and water).