Sequential purification and characterization of Torpedo californica nAChR-DC supplemented with CHS for high-resolution crystallization studies.

Over the past 10 years we have been developing a multi-attribute analytical platform that allows for the preparation of milligram amounts of functional, high-pure, and stable Torpedo (muscle-type) nAChR detergent complexes for crystallization purpose. In the present work, we have been able to significantly improve and optimize the purity and yield of nicotinic acetylcholine receptors in detergent complexes (nAChR-DC) without compromising stability and functionality. We implemented new methods in the process, such as analysis and rapid production of samples for future crystallization preparations. Native nAChR was extracted from the electric organ of Torpedo californica using the lipid-like detergent LysoFos Choline 16 (LFC-16), followed by three consecutive steps of chromatography purification. We evaluated the effect of cholesteryl hemisuccinate (CHS) supplementation during the affinity purification steps of nAChR-LFC-16 in terms of receptor secondary structure, stability and functionality. CHS produced significant changes in the degree of ß-secondary structure, these changes compromise the diffusion of the nAChR-LFC-16 in lipid cubic phase. The behavior was reversed by Methyl-ß-Cyclodextrin treatment. Also, CHS decreased acetylcholine evoked currents of Xenopus leavis oocyte injected with nAChR-LFC-16 in a concentration-dependent manner. Methyl-ß-Cyclodextrin treatment do not reverse functionality, however column delipidation produced a functional protein similar to nAChR-LFC-16 without CHS treatment.

Different metabolism of EPA, DPA and DHA in humans: A double-blind cross-over study.

This study aimed to compare eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) incorporated into red blood cells (RBC) phospholipids (PL), plasma PL, plasma triglyceride (TAG), and plasma cholesteryl ester (CE) fractions, and the metabolomics profiles in a double-blind cross-over study. Twelve female healthy subjects randomly consumed 1 g per day for 6 days of pure EPA, DPA, or DHA. The placebo treatment was olive oil. The fasting venous blood was taken at days 0, 3 and 6, and the RBC PL and plasma lipid fractions were separated for fatty acid determination using thin layer chromatography followed by gas chromatography. Plasma metabolites were analyzed by UHPLC-Q-Exactive Orbitrap/MS. Supplemental EPA significantly increased the concentrations of EPA in RBC PL (days 3 and 6). For subjects consuming the DPA supplement, the concentrations of both DPA and EPA were significantly increased in RBC PL over a 6-day period, respectively. For plasma PL fraction, EPA and DPA supplementation significantly increased the concentrations of EPA and DPA at both days 3 and 6, respectively. Supplemental DHA significantly increased the concentrations of DHA in plasma PL at day 6. For plasma TAG fraction, supplementation with EPA and DPA significantly increased the concentrations of EPA and DPA at both days 3 and 6, respectively. After DHA supplementation, significant increases in the concentrations of DHA were found relative to baseline at both days 3 and 6. For plasma CE fraction, EPA supplementation significantly increased the concentrations of EPA (days 3 and 6) and DPA (days 6), respectively. Supplemental DPA significantly increased the concentrations of EPA at day 6. Meanwhile, the concentrations of DHA were significantly increased over a 6-day period of intervention after subjects consuming the DHA supplements. There were a total of 922 plasma metabolites identified using metabolomics analyses. Supplementation with DPA and DHA significantly increased the levels of sphingosine 1-phosphate (P for DPA = 0.025, P for DHA = 0.029) and 15-deoxy-Δ12,14-prostaglandin A1 (P for DPA = 0.034; P for DHA = 0.021) in comparison with olive oil group. Additionally, supplementation with EPA (P = 0.007) and DHA (P = 0.005) significantly reduced the levels of linoleyl carnitine, compared with olive oil group. This study shows that DPA might act as a reservoir of n-3 LCP incorporated into blood lipid fractions, metabolized into DHA, and retro-converted back to EPA. Metabolomics analyses indicate that supplemental EPA, DPA and DHA have shared and differentiated metabolites. The differences of these metabolic biomarkers should be investigated in additional studies.

Anti-Inflammatory Effects of 5α,8α-Epidioxycholest-6-en-3ß-ol, a Steroidal Endoperoxide Isolated from Aplysia depilans, Based on Bioguided Fractionation and NMR Analysis.

Sea hares of Aplysia genus are recognized as a source of a diverse range of metabolites. 5α,8α-Endoperoxides belong to a group of oxidized sterols commonly found in marine organisms and display several bioactivities, including antimicrobial, anti-tumor, and immunomodulatory properties. Herein we report the isolation of 5α,8α-epidioxycholest-6-en-3ß-ol (EnP(5,8)) from Aplysia depilans Gmelin, based on bioguided fractionation and nuclear magnetic resonance (NMR) analysis, as well as the first disclosure of its anti-inflammatory properties. EnP(5,8) revealed capacity to decrease cellular nitric oxide (NO) levels in RAW 264.7 macrophages treated with lipopolysaccharide (LPS) by downregulation of the Nos2 (inducible nitric oxide synthase, iNOS) gene. Moreover, EnP(5,8) also inhibited the LPS-induced expression of cyclooxygenase-2 (COX-2), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) at the mRNA and protein levels. Mild selective inhibition of COX-2 enzyme activity was also evidenced. Our findings provide evidence of EnP(5,8) as a potential lead drug molecule for the development of new anti-inflammatory agents.

Hexadecenoic Fatty Acid Positional Isomers and De Novo PUFA Synthesis in Colon Cancer Cells.

Palmitic acid metabolism involves delta-9 and delta-6 desaturase enzymes forming palmitoleic acid (9cis-16:1; n-7 series) and sapienic acid (6cis-16:1; n-10 series), respectively. The corresponding biological consequences and lipidomic research on these positional monounsaturated fatty acid (MUFA) isomers are under development. Furthermore, sapienic acid can bring to the de novo synthesis of the n-10 polyunsaturated fatty acid (PUFA) sebaleic acid (5cis,8cis-18:2), but such transformations in cancer cells are not known. The model of Caco-2 cell line was used to monitor sapienic acid supplementation (150 and 300 µM) and provide evidence of the formation of n-10 fatty acids as well as their incorporation at levels of membrane phospholipids and triglycerides. Comparison with palmitoleic and palmitic acids evidenced that lipid remodelling was influenced by the type of fatty acid and positional isomer, with an increase of 8cis-18:1, n-10 PUFA and a decrease of saturated fats in case of sapienic acid. Cholesteryl esters were formed only in cases with sapienic acid. Sapienic acid was the less toxic among the tested fatty acids, showing the highest EC50s and inducing death only in 75% of cells at the highest concentration tested. Two-photon fluorescent microscopy with Laurdan as a fluorescent dye provided information on membrane fluidity, highlighting that sapienic acid increases the distribution of fluid regions, probably connected with the formation of 8cis-18:1 and the n-10 PUFA in cell lipidome. Our results bring evidence for MUFA positional isomers and de novo PUFA synthesis for developing lipidomic analysis and cancer research.

Complete 1H NMR assignment of cholesteryl benzoate.

The 750 MHz 1H NMR spectrum of cholesteryl benzoate (1b) could be assigned completely, which means all chemical shifts and all coupling constants, including some long-range values, were established. This task was possible by extracting many approximate coupling constant values in the overlapped spectrum region from an HSQC experiment, and using these values in the 1H iterative full spin analysis integrated in the PERCH NMR software. The task was facilitated using our published data for 3ß-acetoxypregna-5,16-dien-20-one (3), the assignment data of the sesquiterpene benzoquinone dihydroperezone (2), also performed in the present study, which contains the same carbon atoms chain than cholesterol (1a), and an HSQC study of (25R)-27-deuteriocholesterol (1c) we prepared some 40 years ago. The HSQC values of 1c in combination with the coupling constants of 1b also allowed to completely assigning the spectrum of 1c. The complete assignment of 1b and 1c further provided the opportunity to estimate the hydrogen shifts induced upon benzoylation of cholesterol. Comparison of the experimental vicinal coupling constants of 1b with the values calculated using the Altona software provides an excellent correlation. In addition, a single crystal X-ray diffraction study of 1b provided the molecular conformation in the solid state, which revealed the side chain adopts an extended conformation.

Creation and study of triterpenoid nanoparticles and radioprotective substance genistein.

This work is devoted to the study and obtaining of new radioprotective agents based on natural flavonoid genistein and spherical amorphous nanoparticles (SANPs) produced from a mixture of birch bark triterpenoids. The physicochemical characteristics of the nanoparticles were studied by electron microscopy, dynamic light scattering, and UV-VIS spectroscopy. The radioprotective efficacy of the nanodrug in vivo and the possibility of its use as a radioprotective agent was shown.

Metabolic Conversion of Ceramides in HeLa Cells - A Cholesteryl Phosphocholine Delivery Approach.

Ceramides can be delivered to cultured cells without solvents in the form of complexes with cholesteryl phosphocholine. We have analysed the delivery of three different radiolabeled D-erythro-ceramides (C6-Cer, C10-Cer and C16-Cer) to HeLa cells, and followed their metabolism as well as the cell viability. We found that all three ceramides were successfully taken up by HeLa cells when complexed to CholPC in an equimolar ratio, and show that the ceramides show different rates of cellular uptake and metabolic fate. The C6-Cer had the highest incorporation rate, followed by C10-Cer and C16-Cer, respectively. The subsequent effect on cell viability strongly correlated with the rate of incorporation, where C6-Cer had the strongest apoptotic effects. Low-dose (1 µM) treatment with C6-Cer favoured conversion of the precursor to sphingomyelin, whereas higher concentrations (25-100 µM) yielded increased conversion to C6-glucosylceramide. Similar results were obtained for C10-Cer. In the lower-dose C16-Cer experiments, most of the precursor was degraded, whereas at high-dose concentrations the precursor remained un-metabolized. Using this method, we demonstrate that ceramides with different chain lengths clearly exhibit varying rates of cellular uptake. The cellular fate of the externally delivered ceramides are clearly connected to their rate of incorporation and their subsequent effects on cell viability may be in part determined by their chain length.

Polyunsaturated Fat Intake Estimated by Circulating Biomarkers and Risk of Cardiovascular Disease and All-Cause Mortality in a Population-Based Cohort of 60-Year-Old Men and Women.

BACKGROUND: High intake of polyunsaturated fatty acids (PUFAs) may reduce the risk of cardiovascular disease (CVD) and mortality. Large, prospective studies including both sexes and circulating PUFAs as dietary biomarkers are needed. We investigated sex-specific associations of the major dietary PUFAs, eicosapentaenoic acid, docohexaenoic acid, linoleic acid, and α-linolenic acid, with incident CVD and all-cause mortality in a population-based cohort. METHODS AND RESULTS: PUFAs in serum cholesterol esters were measured at baseline in 60-year-old Swedish women (n=2193) and men (n=2039). With the use of national registers, 484 incident CVD events (294 men and 190 women) and 456 all-cause deaths (265 men and 191 women) were identified during follow-up (median, 14.5 years) in individuals without prior CVD at baseline. Associations of PUFAs with CVD and mortality were evaluated with Cox proportional hazard models. In multivariable-adjusted models, 1-SD increases in eicosapentaenoic acid and docohexaenoic acid were associated with lower risk of incident CVD among women (hazard ratio [HR], 0.79 [95% confidence interval (CI), 0.64-0.97] and 0.74 [95% CI, 0.61-0.89], respectively). α-Linolenic acid was associated with moderately increased CVD risk in women (HR, 1.16; 95% CI, 1.02-1.32). Inverse associations with all-cause mortality were observed for eicosapentaenoic acid and docohexaenoic acid among all participants (HR, 0.81 [95% CI, 0.72-0.91] and 0.80 [95% CI, 0.72-0.89], respectively) and for linoleic acid in men (HR, 0.73; 95% CI, 0.64-0.83). CONCLUSIONS: Serum linoleic acid and very-long-chain n-3 PUFAs, partly reflecting vegetable oil and fish intake, respectively, were inversely associated with all-cause mortality. Inverse associations of eicosapentaenoic acid and docohexaenoic acid with incident CVD were observed only in women.

Cholesteryl-modification of a glucomannan from Bletilla striata and its hydrogel properties.

A glucomannan-type polysaccharide, named BSP, was obtained from the tubers of Bletilla striata by ultrasonic-assisted extraction, ethanol precipitation, deproteination and gel-permeation chromatography. HPLC analysis revealed that BSP contained mannose and glucose in the molar ratio of 3.5:1. Its molecular weight (Mw) was estimated to be 20 kDa. Methylation analysis, FT-IR and NMR analyses indicated that BSP consisted of (1→4)-linked ß-D-glucopyranosyl residues and (1→4)-linked ß-D-mannopyranosyl residues. Cholesteryl succinate was linked to BSP to make it more amphiphilic and the degree of substitution of cholesteryl succinate-BSP was 3.2%. The critical micelle concentration of modified BSP was 0.001 mg/mL, suggesting it could self-assemble into nanoparticles in aqueous solution.

In vivo metabolic fingerprinting of neutral lipids with hyperspectral stimulated Raman scattering microscopy.

Metabolic fingerprinting provides valuable information on the physiopathological states of cells and tissues. Traditional imaging mass spectrometry and magnetic resonance imaging are unable to probe the spatial-temporal dynamics of metabolites at the subcellular level due to either lack of spatial resolution or inability to perform live cell imaging. Here we report a complementary metabolic imaging technique that is based on hyperspectral stimulated Raman scattering (hsSRS). We demonstrated the use of hsSRS imaging in quantifying two major neutral lipids: cholesteryl ester and triacylglycerol in cells and tissues. Our imaging results revealed previously unknown changes of lipid composition associated with obesity and steatohepatitis. We further used stable-isotope labeling to trace the metabolic dynamics of fatty acids in live cells and live Caenorhabditis elegans with hsSRS imaging. We found that unsaturated fatty acid has preferential uptake into lipid storage while saturated fatty acid exhibits toxicity in hepatic cells. Simultaneous metabolic fingerprinting of deuterium-labeled saturated and unsaturated fatty acids in living C. elegans revealed that there is a lack of interaction between the two, unlike previously hypothesized. Our findings provide new approaches for metabolic tracing of neutral lipids and their precursors in living cells and organisms, and could potentially serve as a general approach for metabolic fingerprinting of other metabolites.

Synthesis and properties of cholesteryl 4-(Alkanoylamino)benzoates: liquid crystals and organogelators.

As a new liquid crystal and organogelator, cholesteryl 4-(alkanoylamino)benzoates were prepared. Cholesteryl 4-(alkanoylamino)benzoates had enantiotropic cholesteric and chiral smectic C phases. Furthermore cholesteryl 4-(alkanoylamino)benzoates gelled organic liquid such as 1-decanol, linalool, geraniol, nerol, citronellol, linalyl acetate, lavender oil, orange oil, and rose oil. The terpene and perfume gels show good release characteristics of the volatile components for a long period.

Cancer death is related to high palmitoleic acid in serum and to polymorphisms in the SCD-1 gene in healthy Swedish men.

BACKGROUND: A high proportion of monounsaturated fatty acids (MUFAs) or a high ratio of MUFAs to saturated fatty acids in plasma, reflecting a high activity of the lipogenic enzyme stearoyl-CoA desaturase-1 (SCD-1), has been shown to be related to cancer death and incidence in some studies. OBJECTIVES: The objective was to study whether the serum cholesteryl ester proportion of palmitoleic acid [16:1n-7 (16:1ω-3)] and the ratio of palmitoleic to palmitic acid (16:1n-7/16:0), as an estimation of the activity of SCD-1, are related to cancer death and to investigate whether polymorphisms in the SCD-1 gene are related to cancer mortality. DESIGN: A community-based cohort of 50-y-old men was followed for a maximum of >40 y. Survival analysis was used to relate fatty acid composition in serum, analyzed at baseline by gas-liquid chromatography (n = 1981), and single nucleotide polymorphisms in the SCD-1 gene (n = 986) to cancer death. A 7-d dietary record was completed at age 70 y (n = 880). RESULTS: The proportions of 16:1n-7 and the ratio of 16:1n-7 to 16:0 were associated with cancer mortality during follow-up in a comparison of the highest with the lowest quartile of 16:1n-7 (adjusted HR: 1.37; 95% CI: 1.04, 1.82). Inherited variance of the SCD-1 gene seemed to be related to cancer death, especially among men with a low proportion of PUFA in the diet in a comparison of the highest with the lowest weighted genetic risk score (HR: 2.14; 95% CI: 1.13, 4.04). CONCLUSION: The findings are compatible with the hypothesis that there is an association between endogenously synthesized MUFAs and cancer death.

New applications of phospholipid vesicle-based permeation assay: permeation model mimicking skin barrier.

The phospholipid vesicle-based permeation assay (PVPA), based on a tight barrier composed of liposomes mimicking cells, is providing an opportunity to predict passive drug permeability through biological membranes. Although it was originally developed to mimic the intestinal epithelia, this study focuses on its potential as a simple and affordable skin model for transdermal permeation of drug candidates and evaluation of various drugs and formulations at an early development stage. The changes induced in lipid composition of the lipid-based barriers to better mimic the in vivo stratum corneum lipid composition required optimization of liposomal properties and manufacturing conditions applied in barrier formation. The preparation conditions could be modified to prepare lipid-based barriers of different degrees of leakiness, potentially representing different degree of intact and compromised skin. The different PVPA models developed in this study appeared to be able to distinguish between drugs with different degrees of lipophilicity and penetration potential. Moreover, the PVPA can be produced in controlled and reproducible manner with different degree of leakiness. The model could therefore be applied in both pharmaceutical and cosmeceuticals manufacturing and also has the potential to provide deeper insight on safety of nanodelivery systems administered onto the skin.

A short-term n-3 DPA supplementation study in humans.

PURPOSE: Despite the detailed knowledge of the absorption and incorporation of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) into plasma lipids and red blood cells (RBC) in humans, very little is known about docosapentaenoic acid (DPA, 22:5 n-3). The aim of this study was to investigate the uptake and incorporation of pure DPA and EPA into human plasma and RBC lipids. METHODS: Ten female participants received 8 g of pure DPA or pure EPA in randomized crossover double-blinded manner over a 7-day period. The placebo treatment was olive oil. Blood samples were collected at days zero, four and seven, following which the plasma and RBC were separated and used for the analysis of fatty acids. RESULTS: Supplementation with DPA significantly increased the proportions of DPA in the plasma phospholipids (PL) (by twofold) and triacylglycerol (TAG) fractions (by 2.3-fold, day 4). DPA supplementation also significantly increased the proportions of EPA in TAG (by 3.1-fold, day 4) and cholesterol ester (CE) fractions (by 2.0-fold, day 7) and of DHA in TAG fraction (by 3.1-fold, day 4). DPA proportions in RBC PL did not change following supplementation. Supplementation with EPA significantly increased the proportion of EPA in the plasma CE and PL fractions, (both by 2.7-fold, day 4 and day 7) and in the RBC PL (by 1.9-fold, day 4 and day 7). EPA supplementation did not alter the proportions of DPA or DHA in any lipid fraction. These results showed that within day 4 of supplementation, DPA and EPA demonstrated different and specific incorporation patterns. CONCLUSION: The results of this short-term study suggest that DPA may act as a reservoir of the major long-chain n-3 fatty acids (LC n-3 PUFA) in humans.

Do lipids retard the evaporation of the tear fluid?

PURPOSE: We examined in vitro the potential evaporation-retarding effect of the tear film lipid layer (TFLL). The artificial TFLL compositions used here were based on the present knowledge of TFLL composition. METHODS: A custom-built system was developed to measure evaporation rates at 35°C. Lipids were applied to an air-water interface, and the evaporation rate through the lipid layer was defined as water loss from the interface. A thick layer of olive oil and a monolayer of long-chain alcohol were used as controls. The artificial TFLLs were composed of 1 to 4 lipid species: polar phosphatidylcholine (PC), nonpolar cholesteryl ester, triglycerides, and wax ester (WE). Brewster angle microscopy (BAM) and interfacial shear rheometry (ISR) were used to assess the lateral structure and shear stress response of the lipid layers, respectively. RESULTS: Olive oil and long-chain alcohol decreased evaporation by 54% and 45%, respectively. The PC monolayer and the four-component mixtures did not retard evaporation. WE was the most important evaporation-retardant TFLL lipid (∼20% decrease). In PC/WE mixtures, an ∼90% proportion of WE was required for evaporation retardation. Based on BAM and ISR, WE resulted in more condensed layers than the non-retardant layers. CONCLUSIONS: Highly condensed, solid-like lipid layers, such as those containing high proportions of WEs, are evaporation-retardant. In multi-component lipid layers, the evaporation-retardant interactions between carbon chains decrease and, therefore, these lipid layers do not retard evaporation.

Study of the miscibility of cholesteryl oleate in a matrix of ceramide, cholesterol and fatty acid.

Cholesteryl esters (CE) are not generally abundant but are ubiquitous in living organisms and have markedly different properties from cholesterol because of their acyl chain. The miscibility/immiscibility of CE with biological lipid structures is a key property for their functions. In this work we study the solubility of cholesteryl oleate (ChO) in a model of the stratum corneum lipid matrix composed of ceramide C16, cholesterol and palmitic acid in excess water. Experiments were done in conditions of fully ionized (pH=9.0) and fully neutralized fatty acid (pH=4.0), and differential scanning calorimetry of the ternary mixtures with added ChO at pH=9.0 clearly displayed a main transition with the same maximum temperature, peak shape, and enthalpy, suggesting that ChO was excluded from the remaining lipids. This technique is not conclusive at pH=4.0 because the transitions of the lipid matrix and ChO overlap. The insolubility of ChO at both pH values is supported by X-ray diffraction. Adding the ceramide:cholesterol:fatty acid lipid mixture to ChO did not change the X-ray pattern of the mixture nor that of the ChO. To supplement the above physical techniques, we applied (13)C MAS NMR spectroscopy with C-13 carbonyl-labeled ChO. A single (13)C carbonyl peak from the ChO at 171.5 ppm was observed, indicating exposure to only one environment. The chemical shift was identical to pure ChO below and above the temperature of isotropic liquid formation. Taken together, our results lead to the conclusion that the solubility of ChO is negligible in the ceramide:cholesterol:fatty acid lipid mixture.

The physical state of lipid nanoparticles influences their effect on in vitro cell viability.

Although lipid nanoparticles represent potent drug carriers, for many formulations toxicity data are rare. Thus, in this study, the effect of different lipid nanoparticles on the cell viability of L929 mouse fibroblasts was systematically investigated using the MTT assay. The formulations were composed of trimyristin, tristearin or cholesteryl myristate stabilized with poloxamer 188, polysorbate 80, polyvinyl alcohol or a blend of soybean phospholipid and sodium glycocholate. Depending on lipid and storage conditions, the nanoparticles were prepared in different physical states or crystal modifications leading to different particle shapes. The cell viability was influenced considerably by the physical state of the particle matrix with crystalline nanoparticles causing a stronger decrease in viability than the corresponding liquid or liquid crystalline particles. Effects on the cell viability were also related to the type of matrix lipid, stabilizer and the particle shape. However, the effects of differently shaped particles of different polymorphic modifications of crystalline tristearin were comparable. The low viability caused by poloxamer 188-stabilized particles could be correlated with a strong cell uptake which was investigated by confocal laser scanning microscopy.

The effect of fatty or lean fish intake on inflammatory gene expression in peripheral blood mononuclear cells of patients with coronary heart disease.

BACKGROUND: Little is known about the effect of fish consumption on gene expression of inflammation-related genes in immune cells in coronary heart disease (CHD). AIM OF THE STUDY: We sought to evaluate the effect of a fatty fish (FF) or a lean fish (LF) diet on the modulation of inflammatory and endothelial function-related genes in peripheral blood mononuclear cells (PBMCs) of subjects with CHD, and its association with serum fatty acid (FA) profile and lipid metabolic compounds. METHODS: Data from 27 patients randomized into an 8-week FF (n = 10; mean +/- SD: 4.3 +/- 0.4 portions of fish per week), LF (n = 11; 4.7 +/- 1.1 portions of fish per week), or control diet (n = 6; 0.6 +/- 0.4 portions of fish per week) were analyzed. The mRNA expression was measured using real-time PCR. RESULTS: The effect of the intervention on the mRNA expression of the genes studied did not differ among groups. In the FF group, however, the decrease in arachidonic acid to eicosapentaenoic acid (AA:EPA) ratio in cholesterol ester and phospholipid fractions strongly correlated with the change in IL1B mRNA levels (r (s) = 0.60, P = 0.06 and r (s) = 0.86, P = 0.002, respectively). In the LF group, the decrease in palmitic acid and total saturated FAs in cholesterol esters correlated with the change in intercellular cell adhesion molecule-1 (ICAM1) expression (r (s) = 0.64, P = 0.04 for both). Circulating levels of soluble ICAM-1 decreased only in the LF group (P < 0.05). CONCLUSIONS: The intake of FF or LF diet did not alter the expression of inflammatory and endothelial function-related genes in PBMCs of patients with CHD. However, the decrease in AA:EPA ratio in serum lipids in the FF group may induce an anti-inflammatory response at mRNA levels in PBMCs. A LF diet might benefit endothelial function, possibly mediated by the changes in serum FA composition.

Uptake by breast carcinoma of a lipidic nanoemulsion after intralesional injection into the patients: a new strategy for neoadjuvant chemotherapy.

OBJECTIVE: Previously we showed that after intravenous injection a lipidic nanoemulsion concentrates in breast carcinoma tissue and other solid tumors and may carry drugs directed against neoplastic tissues. Use of the nanoemulsion decreases toxicity of the chemotherapeutic agents without decreasing the anticancer action. Currently, the hypothesis was tested whether the nanoemulsion concentrates in breast carcinoma tissue after locoregional injection. METHODS: Three different techniques of injection of the nanoemulsion were tested in patients scheduled for surgical treatment: G1 (n=4) into the mammary tissue 5 cm away from the tumor; G2 (n=4) into the peritumoral mammary tissue; G3 (n=6) into the tumoral tissue. The nanoemulsion labeled with radioactive cholesteryl oleate was injected 12 h before surgery; plasma decay of the label was determined from blood samples collected over 24 h and the tissue fragments excised during the surgery were analyzed for radioactivity uptake. RESULTS: Among the three nanoemulsion injection techniques, G3 showed the greatest uptake (data expressed in c.p.m/g of tissue) by the tumor (44,769+/-54,749) and by the lymph node (2356+/-2966), as well as the greatest concentration in tumor compared to normal tissue (844+/-1673). In G1 and G2, uptakes were, respectively, tumor: 60+/-71 and 843+/-1526; lymph node: 263+/-375 and 102+/-74; normal tissue: 139+/-102 and 217+/-413. CONCLUSIONS: Therefore, with intralesional injection of the nanoemulsion, a great concentration effect can be achieved. This injection technique may be thus a promising approach for drug-targeting in neoadjuvant chemotherapy in breast cancer treatment.

Novel designed probes for the characterization of oxidative stress in biological fluids, cells, and tissues.

Oxidative stress (OS) is linked to the development of human diseases. Early identification of OS-associated diseases is essential in the control of their progression and treatment. Efforts have been undertaken to identify reliable endogenous markers, which correlate with the progression of a disease in an organ undergoing OS. An ideal biomarker must be validated, utilize noninvasive sampling, and have a simple, specific and highly sensitive detection method. Among the currently used markers assessing OS, are those that are nonspecific (peroxide value [PV], conjugated dienes [CD], thiobarbitoric acid reactive substances [TBARS]), and others that measure end-products of oxidized degradation biomolecules (isoprostanes, oxysterols, keto-proteins, 8-oxodeoxyguanosine), whose accumulation is not necessarily correlated with augmented OS. The search for a more reliable marker necessitates new approaches to fulfill such requirements and overcome many of the obstacles associated with the current markers. We suggest a new strategy of using designed exogenous novel reporters, constructed from endogenous subunits, that are sensitive to reactive oxygen and nitrogen species (ROS/RNS) and commonly known to react with them, forming specific oxidized products. These subunits are tyrosine (representing proteins), bonded covalently to linoleic acid (representing polyunsaturated fatty acids) forming an amide bond, which can be further connected through an ester bond to a third unit, either to cholesterol (representing sterols) or to 2'-deoxyguanosine (representing DNA). Oxidation of the designed probe can outline, in real time, the formation of oxidation products and distinguish them from intrinsic biomolecules, provide information about the relative subunit susceptibilities to a specific oxidant challenge, and allow for the assessment of the utility of intervention, such as antioxidant supplementation. By utilizing such markers, it may be possible to correlate between the damaged fingerprints of the marker and the specific pathological conditions. The above markers were tested to characterize OS in in vitro and in in vivo experiments, such as in those carried out in human fluids (blood, serum, saliva), tissues (brain or muscle homogenates), and cells (macrophages, astrocytes, neurons), pertaining to OS-associated diseases, such as atherosclerosis, diabetes, and Alzheimer's disease.