Amaranth protein presents cholesterol-lowering effect

This study describes amaranth`s protein cholesterol-lowering effect and investigates its mechanisms hypercholesterolaemia was induced in male hamsters through diet rich in casein (300 g/kg diet) containing regular levels of cholesterol (0.5 kg/g) fed during 3 weeks. Animals were divided into three groups and fed ad libitum diets for 4 weeks containing as the sole source of protein: casein (control), amaranth protein isolate or, casein + amaranth protein isolate. Plasma concentrations of cholesterol and triacylglycerols were measured at four different points: at the beginning of the study. after hypercholesterolaemia was induced, in the first week and then at the end of the experimental diet period. The reduction of the total plasma cholesterol concentration at the end of experimental period for animals fed on diets containing amaranth protein isolate pure and with casein were 27% (P < 0.05) and 48% (P < 0.05). respectively, being the non-HDL fractions the most affected. Digestibility of protein as well as excretion of cholesterol and bile acid, were investigated as the possible mechanisms for this significant hypocholesterolaemic effect. Cholesterol excretion was related to the hypocholesterolaemia but could not explain all the observed reduction. Our findings suggest that amaranth protein has a metabolic effect on endogenous cholesterol metabolism. (C) 2009 Elsevier Ltd. All rights reserved.

Cholesterol-Lowering Properties of Whole Cowpea Seed and Its Protein Isolate in Hamsters

Hypercholesterolemic hamsters were fed for 4 wk on diets rich in saturated fatty acids and cholesterol, differing only in protein source (20%): casein (control group, HC), whole cowpea seed (HWS), and cowpea protein isolate (HPI). Hamsters fed on HWS and HPI presented significant reductions in plasma total cholesterol and non-HDL cholesterol. HPI and HC presented similar protein digestibility, which were significantly higher than that of HWS. Animals fed on HWS presented significantly higher levels of bile acids and cholesterol in feces than did the animals fed on casein or HPI diets. Histological analyses of the liver showed that HC diet resulted in steatosis widely distributed throughout the hepatic lobule, while HWS and HPI diets promoted reductions in liver steatosis. The effectiveness of HWS for modulating lipid metabolism was greater than that of HPI, as measured by plasma cholesterol reduction and liver steatosis.

Increase of Cholesterol Oxidation and Decrease of PUFA as a Result of Thermal Processing and Storage in Eggs Enriched with n-3 Fatty Acids

In this work, cholesterol oxide formation and alteration of fatty acid composition were analyzed in n-3 enriched eggs under different storage periods and two temperatures. The eggs enriched with n-3 fatty acids were stored at 5 or 25 degrees C for 45 days and subsequently boiled or fried. For each treatment, 12 yolks were analyzed every 15 days including time zero. The concentrations of the cholesterol oxides 7-ketocholesterol, 7 beta-hydroxycholesterol, and 7 alpha-hydroxycholesterol increased during the storage period and were higher in fried eggs. Only the 7-ketocholesterol was affected by the storage temperature, and its concentration was highest in eggs stored at 25 degrees C. There was no significant difference in the contents of cholesterol and vitamin E at the different storage periods; however, the concentration of vitamin E decreased with thermal treatment. In addition, the n-3 polyunsaturated fatty acids, especially 18:3, 20:5, and 22:6, were reduced throughout the storage at 5 and 25 degrees C.

Effects of lipid-lowering drugs on reverse cholesterol transport gene expressions in peripheral blood mononuclear and HepG2 cells

Aims: The ATP-binding cassette transporters, ABCA1 and ABCG1, are LXR-target genes that play an important role in reverse cholesterol transport. We examined the effects of inhibitors of the cholesterol absorption (ezetimibe) and synthesis (statins) on expression of these transporters in HepG2 cells and peripheral blood mononuclear cells (PBMCs) of individuals with primary (and nonfamilial) hypercholesterolemia (HC). Materials & methods: A total of 48 HC individuals were treated with atorvastatin (10 mg/day/4 weeks) and 23 were treated with ezetimibe (10 mg/day/4 weeks), followed by simvastatin (10 mg/day/8 weeks) and simvastatin plus ezetimibe (10 mg of each/day/4 weeks). Gene expression was examined in statin- or ezetimibe-treated and control HepG2 cells as well as PBMCs using real-time PCR. Results: In PBMCs, statins and ezetimibe downregulated ABCA1 and ABCG1 mRNA expression but did not modulate NR1H2 (LxR-beta) and NR1H3 (LXR-alpha) levels. Positive correlations of ABCA1 with ABCG1 and of NR1H2 with NR1H3 expressions were found in all phases of the treatments. In HepG2 cells, ABCA1 mRNA levels remained unaltered while ABCG1 expression was increased by statin (1.0-10.0 mu M) or ezetimibe (5.0 mu M) treatments. Atorvastatin upregulated NR1H2 and NR1H3 only at 10.0 mu M, meanwhile ezetimibe (1.0-5.0 mu M) downregulated NR1H2 but did not change NR1H3 expression. Conclusion: Our findings reveal that lipid-lowering drugs downregulate ABCA1 and ABCG1 mRNA expression in PBMCs of HC individuals and exhibit differential effects on HepG2 cells. Moreover, they indicate that the ABCA1 and ABCG1 transcript levels were not correlated directly to LXR mRNA expression in both cell models treated with lipid-lowering drugs.

Laurdan Spectrum Decomposition as a Tool for the Analysis of Surface Bilayer Structure and Polarity: a Study with DMPG, Peptides and Cholesterol

The highly hydrophobic fluorophore Laurdan (6-dodecanoyl-2-(dimethylaminonaphthalene)) has been widely used as a fluorescent probe to monitor lipid membranes. Actually, it monitors the structure and polarity of the bilayer surface, where its fluorescent moiety is supposed to reside. The present paper discusses the high sensitivity of Laurdan fluorescence through the decomposition of its emission spectrum into two Gaussian bands, which correspond to emissions from two different excited states, one more solvent relaxed than the other. It will be shown that the analysis of the area fraction of each band is more sensitive to bilayer structural changes than the largely used parameter called Generalized Polarization, possibly because the latter does not completely separate the fluorescence emission from the two different excited states of Laurdan. Moreover, it will be shown that this decomposition should be done with the spectrum as a function of energy, and not wavelength. Due to the presence of the two emission bands in Laurdan spectrum, fluorescence anisotropy should be measured around 480 nm, to be able to monitor the fluorescence emission from one excited state only, the solvent relaxed state. Laurdan will be used to monitor the complex structure of the anionic phospholipid DMPG (dimyristoyl phosphatidylglycerol) at different ionic strengths, and the alterations caused on gel and fluid membranes due to the interaction of cationic peptides and cholesterol. Analyzing both the emission spectrum decomposition and anisotropy it was possible to distinguish between effects on the packing and on the hydration of the lipid membrane surface. It could be clearly detected that a more potent analog of the melanotropic hormone alpha-MSH (Ac-Ser(1)-Tyr(2)-Ser(3)-Met(4)-Glu(5)-His(6)-Phe(7)-Arg(8)-Trp(9)-Gly(10)-Lys(11)-Pro(12)-Val(13)-NH(2)) was more effective in rigidifying the bilayer surface of fluid membranes than the hormone, though the hormone significantly decreases the bilayer surface hydration.

Cholesterol Mediates Chitosan Activity on Phospholipid Monolayers and Langmuir-Blodgett Films

The polysaccharide chitosan has been largely used in many biological applications as a fat and cholesterol reducer, bactericide agent, and wound healing material. While the efficacy for some of such uses is proven, little is known about the molecular-level interactions involved in these applications. In this study, we employ mixed Langmuir and Langmuir-Blodgett (LB) films of negatively charged dimyristoyl phosphatidic acid (DMPA) anti cholesterol as cell membrane models to investigate the role of cholesterol in the molecular-level action of chitosan. Chitosan does not remove cholesterol froth the monolayer. The interaction with chitosan tends to expand the DMPA monolayer due to its interpenetration within the film. On the other hand, cholesterol induces condensation of the DMPA monolayer. The competing effects cause the surface pressure isotherms of mixed DMPA-cholesterol films on a chitosan subphase to be unaffected by the cholesterol mole fraction, due to distinct degrees of chitosan penetration into the film in the presence of cholesterol. By combining polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) and sum-frequency generation spectroscopy (SFG), we showed that chitosan induces order into negatively charged phospholipid layers, whereas the opposite occurs for cholesterol. In conclusion, chitosan has its penetration in the film modulated by cholesterol, and electrostatic interactions with negatively charged phospholipids, such as DMPA, are crucial for the action of chitosan.

A reduction of CETP activity, not an increase, is associated with modestly impaired postprandial lipemia and increased HDL-Cholesterol in adult asymptomatic women

Background: The relationship between CETP and postprandial hyperlipemia is still unclear. We verified the effects of varying activities of plasma CETP on postprandial lipemia and precocious atherosclerosis in asymptomatic adult women. Methods: Twenty-eight women, selected from a healthy population sample (n = 148) were classified according to three CETP levels, all statistically different: CETP deficiency (CETPd <= 4.5%, n = 8), high activity (CETPi >= 23.8, n = 6) and controls (CTL, CETP >= 4.6% and <= 23.7%, n = 14). After a 12 h fast they underwent an oral fat tolerance test (40 g of fat/m(2) of body surface area) for 8 hours. TG, TG-rich-lipoproteins (TRL), cholesterol and TRL-TG measurements (AUC, AUIC, AR, RR and late peaks) and comparisons were performed on all time points. Lipases and phospholipids transfer protein (PLTP) were determined. Correlation between carotid atherosclerosis (c-IMT) and postprandial parameters was determined. CETP TaqIB and I405V and ApoE-epsilon 3/epsilon 2/epsilon 4 polymorphisms were examined. To elucidate the regulation of increased lipemia in CETPd a multiple linear regression analysis was performed. Results: In the CETPi and CTL groups, CETP activity was respectively 9 and 5.3 higher compared to the CETPd group. Concentrations of all HDL fractions and ApoA-I were higher in the CETPd group and clearance was delayed, as demonstrated by modified lipemia parameters (AUC, AUIC, RR, AR and late peaks and meal response patterns). LPL or HL deficiencies were not observed. No genetic determinants of CETP deficiency or of postprandial lipemia were found. Correlations with c-IMT in the CETPd group indicated postprandial pro-atherogenic associations. In CETPd the regression multivariate analysis (model A) showed that CETP was largely and negatively predicted by VLDL-C lipemia (R(2) = 92%) and much less by TG, LDL-C, ApoAI, phospholipids and non-HDL-C. CETP (model B) influenced mainly the increment in ApoB-100 containing lipoproteins (R(2) = 85% negatively) and phospholipids (R(2) = 13%), at the 6(th)h point. Conclusion: The moderate CETP deficiency phenotype included a paradoxically high HDL-C and its sub fractions (as earlier described), positive associations with c-IMT, a postprandial VLDL-C increment predicting negatively CETP activity and CETP activity regulating inversely the increment in ApoB100-containing lipoproteins. We hypothesize that the enrichment of TG content in triglyceride-rich ApoB-containing lipoproteins and in TG rich remnants increases lipoproteins` competition to active lipolysis sites, reducing their catabolism and resulting on postprandial lipemia with atherogenic consequences.

Generation of Cholesterol Carboxyaldehyde by the Reaction of Singlet Molecular Oxygen [O(2) ((1)Delta(g))] as Well as Ozone with Cholesterol

A few years ago, it was reported that ozone is produced in human atherosclerotic arteries, on the basis of the identification of 3 beta-hydroxy-5-oxo-5,6-secocholestan-6-al and 3 beta-hydroxy-5 beta-hydroxy-B-norcholestane-6 beta-carboxaldehyde (ChAld) as their 2,4-dinitrophenylhydrazones. The formation of endogenous ozone was attributed to water oxidation catalyzed by antibodies, with the formation of dihydrogen trioxide as a key intermediate. We now report that ChAld is also generated by the reaction of cholesterol with singlet molecular oxygen [O(2) ((1)Delta(g))] that is produced by photodynamic action or by the thermodecomposition of 1,4-dimethylnaphthalene endoperoxide, a defined pure chemical source of O(2) ((1)Delta(g)). On the basis of (18)O-labeled ChAld mass spectrometry, NMR, light emission measurements, and derivatization studies, we propose that the mechanism of ChAld generation involves the formation of the well-known cholesterol 5 alpha-hydroperoxide (5 alpha-OOH) (the major product of O(2) ((1)Delta(g))-oxidation of cholesterol) and/or a 1,2-dioxetane intermediate formed by O(2) ((1)Delta(g)) attack at the Delta(5) position. The Hock cleavage of 5 alpha-OOH (the major pathway) or unstable cholesterol dioxetane decomposition (a minor pathway, traces) gives a 5,6-secosterol intermediate, which undergoes intramolecular aldolization to yield ChAld. These results show clearly and unequivocally that ChAld is generated upon the reaction of cholesterol with O(2) ((1)Delta(g)) and raises questions about the role of ozone in biological processes.

Effect of cholesterol on the interaction of the amphibian antimicrobial peptide DD K with liposomes

DD K is an antimicrobial peptide previously isolated from the skin of the amphibian Phyllomedusa distincta. The effect of cholesterol on synthetic DD K binding to egg lecithin liposomes was investigated by intrinsic fluorescence of tryptophan residue, measurements of kinetics of 5(6)-carboxyfluorescein (CF) leakage, dynamic light scattering and isothermal titration microcalorimetry. An 8 nm blue shift of tryptophan maximum emission fluorescence was observed when DD K was in the presence of lecithin liposomes compared to the value observed for liposomes containing 43 mol% cholesterol. The rate and the extent of CF release were also significantly reduced by the presence of cholesterol. Dynamic light scattering showed that lecithin liposome size increase from 115 to 140 nm when titrated with DD K but addition of cholesterol reduces the liposome size increments. Isothermal titration microcalorimetry studies showed that DD K binding both to liposomes containing cholesterol as to liposomes devoid of it is more entropically than enthalpically favored. Nevertheless, the peptide concentration necessary to furnish an adjustable titration curve is much higher for liposomes containing cholesterol at 43 mol% (2 mmol L-1) than in its absence (93 mu mol L-1). Apparent binding constant values were 2160 and 10,000 L mol(-1), respectively. The whole data indicate that DD K binding to phosphatidylcholine liposomes is significantly affected by cholesterol, which contributes to explain the low hemolytic activity of the peptide. (C) 2007 Elsevier Inc. All rights reserved.

Cholesterol Hydroperoxides Generate Singlet Molecular Oxygen [O(2) ((1)Delta(g))]: Near-IR Emission, (18)O-Labeled Hydroperoxides, and Mass Spectrometry

In mammalian membranes, cholesterol is concentrated in lipid rafts. The generation of cholesterol hydroperoxides (ChOOHs) and their decomposition products induces various types of cell damage. The decomposition of some organic hydroperoxides into peroxyl radicals is known to be a potential source of singlet molecular oxygen [O(2) ((1)Delta(g))] in biological systems. We report herein on evidence of the generation of O(2) ((1)Delta(g)) from ChOOH isomers in solution or in liposomes containing ChOOHs, which involves a cyclic mechanism from a linear tetraoxide intermediate originally proposed by Russell. Characteristic light emission at 1270 nm, corresponding to O(2) ((1)Delta(g)) monomolecular decay, was observed for each ChOOH isomer or in liposomes containing ChOOHs. Moreover, the presence of O(2) ((1)Delta(g)) was unequivocally demonstrated using the direct spectral characterization of near-infrared light emission. Using (18)O-labeled cholesterol hydroperoxide (Ch(18)O(18)OH), we observed the formation of (18)O-labeled O(2) ((1)Delta(g)) [(18)O(2) ((1)Delta(g))] by the chemical trapping of (18)O(2) ((1)Delta(g)) with 9,10-diphenylanthracene (DPA) and detected the corresponding (18)O-labeled DPA endoperoxide (DPA(18)O(18)O) and the (18)O-labeled products of the Russell mechanism using high-performance liquid chromatography coupled to tandem mass spectrometry. Photoemission properties and chemical trapping clearly demonstrate that the decomposition of Ch(18)O(18)OH generates (18)O(2) ((1)Delta(g)), which is consistent with the Russell mechanism and points to the involvement of O(2) ((1)Delta(g)) in cholesterol hydroperoxide-mediated cytotoxicity.

DNA strand breaks and base modifications induced by cholesterol hydroperoxides

Cholesterol (Ch) can be oxidized by reactive oxygen species, forming oxidized products such as Ch hydroperoxides (ChOOH). These hydroperoxides can disseminate the peroxidative stress to other cell compartments. In this work, the ability of ChOOH to induce strand breaks and/or base modifications in a plasmid DNA model was evaluated. In addition, HPLC/MS/MS analyses were performed to investigate the formation of 8-oxo-7,8-dihydro-2`-deoxyguanosine (8-oxodGuo) after the incubation of 2`-deoxyguanosine (dGuo) with ChOOH and Cu(2+). In the presence of copper ions, ChOOH induced DNA strand breaks in time and concentration-dependent manners. Purine and pyrimidine base modifications were also observed, as assessed respectively by the treatment with Fpg and Endo III repair enzymes. The detection of 8-oxodGuo by HPLC/MS/MS is in agreement with the dGuo oxidation in plasmid DNA. ChOOH-derived DNA damage adds further support to the role of lipid peroxidation in inducing DNA modifications and mutation.

Detection and Characterization of Cholesterol-Oxidized Products Using HPLC Coupled to Dopant Assisted Atmospheric Pressure Photoionization Tandem Mass Spectrometry

Oxidation of cholesterol (Ch) by a variety of reactive oxygen species gives rise mainly to hydroperoxides and aldehydes. Despite the growing interest in Ch-oxidized products, the detection and characterization of these products is still a matter of concern. In this work, the main Ch-oxidized products, namely, 3 beta-hydroxycholest-5-ene-7 alpha-hydroperoxide (7 alpha-OOH), 3 beta-5 alpha-cholest-6-ene-5-hydroperoxide (5 alpha-OOH), 3 beta-hydroxycholest-4-ene-6 alpha-hydroperoxide (6 alpha-OOH), 3 beta-hydroxycholest-4-ene-6 beta-hydroperoxide (6 beta-OOH), and 3 beta-hydroxy-5 beta-hydroxy-B-norcholestane-6 beta-carboxaldehyde (ChAld), were detected in the same analysis using high-performance liquid chromatography (HPLC) coupled to dopant assisted atmospheric pressure photoionization tandem mass spectrometry. The use of selected reaction monitoring mode (SRM) allowed a sensitive detection of each oxidized product, while the enhanced product ion mode (EPI) helped to improve the confidence of the analyses. Isotopic labeling experiments enabled one to elucidate mechanistic features during fragmentation processes. The characteristic fragmentation pattern of Ch-oxidized products is the consecutive loss of 1120 molecules, yielding cationic fragments at m/z 401, 383, and 365. Homolytic scissions of the peroxide bond are also seen. With (18)O-labeling approach, it was possible to establish a fragmentation order for each isomer. The SRM transitions ratio along with EPI and (18)O-labeled experiments give detailed information about differences for water elimination, allowing a proper discrimination between the isomers:Phis is of special interest considering the emerging role of Ch-oxidized products in the development of diseases.

Highly Sensitive Fluorescent Method for the Detection of Cholesterol Aldehydes Formed by Ozone and Singlet Molecular Oxygen

Cholesterol oxidation gives rise to a mixture of oxidized products. Different types of products are generated according to the reactive species being involved. Recently, attention has been focused on two cholesterol aldehydes, 3 beta-hydroxy-5 beta-hydroxy-B-norcholestane-6 beta-carboxyaldehyde (1a) and 3 beta-hydroxy-5-oxo-5,6-secocholestan-6-al (1b). These aldehydes can be generated by ozone-, as well as by singlet molecular oxygen-mediated cholesterol oxidation. It has been suggested that 1b is preferentially formed by ozone and la is preferentially formed by singlet molecular oxygen. In this study we describe the use of 1-pyrenebutyric hydrazine (PBH) as a fluorescent probe for the detection of cholesterol aldehydes. The formation of the fluorescent adduct between la with PBH was confirmed by HPLC-MS/MS. The fluorescence spectra of PBH did not change upon binding to the aldehyde. Moreover, the derivatization was also effective in the absence of an acidified medium, which is critical to avoid the formation of cholesterol aldehydes through Hock cleavage of 5 alpha-hydroperoxycholesterol. In conclusion, PBH can be used as an efficient fluorescent probe for the detection/quantification of cholesterol aldehydes in biological samples. Its analysis by HPLC coupled to a fluorescent detector provides a sensitive and specific way to quantify cholesterol aldehydes in the low femtomol range.