Water-Soluble Saccharina latissima Polysaccharides and Relation of Their Structural Characteristics with In Vitro Immunostimulatory and Hypocholesterolemic Activities.

Brown macroalgae are an important source of polysaccharides, mainly fucose-containing sulphated polysaccharides (FCSPs), associated with several biological activities. However, the structural diversity and structure-function relationships for their bioactivities are still undisclosed. Thus, the aim of this work was to characterize the chemical structure of water-soluble Saccharina latissima polysaccharides and evaluate their immunostimulatory and hypocholesterolemic activities, helping to pinpoint a structure-activity relationship. Alginate, laminarans (F1, neutral glucose-rich polysaccharides), and two fractions (F2 and F3) of FCSPs (negatively charged) were studied. Whereas F2 is rich in uronic acids (45 mol%) and fucose (29 mol%), F3 is rich in fucose (59 mol%) and galactose (21 mol%). These two fractions of FCSPs showed immunostimulatory activity on B lymphocytes, which could be associated with the presence of sulphate groups. Only F2 exhibited a significant effect in reductions in in vitro cholesterol's bioaccessibility attributed to the sequestration of bile salts. Therefore, S. latissima FCSPs were shown to have potential as immunostimulatory and hypocholesterolemic functional ingredients, where their content in uronic acids and sulphation seem to be relevant for the bioactive and healthy properties.

Effect of Acyl Chain Length on the Rate of Phospholipid Flip-Flop and Intermembrane Transfer.

The rate at which phospholipids equilibrate between different membranes and between the non-polar environments in biological fluids is of high importance in the understanding of biomembrane diversity, as well as in the development of liposomes for drug delivery. In this work, we characterize the rate of insertion into and desorption from POPC bilayers for a homologous series of amphiphiles with the fluorescent NBD group attached to phosphoethanolamines of different acyl chain lengths, NBD-diC n -PE with n = 6, 8, 10, and 12. The rate of translocation between bilayer leaflets was also characterized, providing all the relevant parameters for their interaction with lipid bilayers. The results are complemented with data for NBD-diC14-PE obtained from literature (Abreu et al. Biophys J 87:353-365, 2004; Moreno et al. Biophys J 91:873-881, 2006). The rate of translocation between the POPC leaflets is not dependent on the length of the acyl chains, while this affects strongly the rate of desorption from the bilayer. Insertion in the POPC bilayer is not diffusion controlled showing a significant dependence on the acyl chain length and on temperature. The results obtained are compared with those previously reported for NBD-LysoC14-PE (Sampaio et al. Biophys J 88:4064-4071, 2005), and with the homologous series of single chain amphiphiles NBD-C n (Cardoso et al. J Phys Chem B 114:16337-16346, 2010; J Phys Chem B 115:10098-10108, 2011). This allows the establishment of important relations between the rate constants for interaction with the lipid bilayers and the structural properties of the amphiphiles, namely the total surface and the cross-section of their non-polar region.

Quantification of Cholesterol Solubilized in Dietary Micelles: Dependence on Human Bile Salt Variability and the Presence of Dietary Food Ingredients.

The solubility of cholesterol in bile salt (BS) micelles is important to understand the availability of cholesterol for absorption in the intestinal epithelium and to develop strategies to decrease cholesterol intake from the intestinal lumen. This has been the subject of intense investigation, due to the established relation between the development of diseases such as atherosclerosis and high levels of cholesterol in the blood. In this work we quantify the effect of BS variability on the amount of cholesterol solubilized. The effect of some known hypocholesterolemic agents usually found in the diet is also evaluated, as well as some insight regarding the mechanisms involved. The results show that, depending on the bile salt composition, the average value of sterol per micelle is equal to or lower than 1. The amount of cholesterol solubilized in the BS micelles is essentially equal to its total concentration until the solubility limit is reached. Altogether, this indicates that the maximum cholesterol solubility in the BS micellar solution is the result of saturation of the aqueous phase and depends on the partition coefficient of cholesterol between the aqueous phase and the micellar pseudophase. The effect on cholesterol maximum solubility for several food ingredients usually encountered in the diet was characterized using methodology developed recently by us. This method allows the simultaneous quantification of both cholesterol and food ingredient solubilized in the BS micelles even in the presence of larger aggregates, therefore avoiding their physical separation with possible impacts on the overall equilibrium. The phytosterols stigmasterol and stigmastanol significantly decreased cholesterol solubility with a concomitant reduction in the total amount of sterol solubilized, most pronounced for stigmasterol. Those results point toward coprecipitation being the major cause for the decrease in cholesterol solubilization by the BS micelles. The presence of tocopherol and oleic acid leads to a small decrease in the amount of cholesterol solubilized while palmitic acid slightly increases the solubility of cholesterol. Those dietary food ingredients are completely solubilized by the BS micelles, indicating that the effects on cholesterol solubility are due to changes in the properties of the mixed micelles.

Interaction of Bile Salts with Model Membranes Mimicking the Gastrointestinal Epithelium: A Study by Isothermal Titration Calorimetry.

Bile salts (BS) are biosurfactants synthesized in the liver and secreted into the intestinal lumen where they solubilize cholesterol and other hydrophobic compounds facilitating their gastrointestinal absorption. Partition of BS toward biomembranes is an important step in both processes. Depending on the loading of the secreted BS micelles with endogeneous cholesterol and on the amount of cholesterol from diet, this may lead to the excretion or absorption of cholesterol, from cholesterol-saturated membranes in the liver or to gastrointestinal membranes, respectively. The partition of BS toward the gastrointestinal membranes may also affect the barrier properties of those membranes affecting the permeability for hydrophobic and amphiphilic compounds. Two important parameters in the interaction of the distinct BS with biomembranes are their partition coefficient and the rate of diffusion through the membrane. Altogether, they allow the calculation of BS local concentrations in the membrane as well as their asymmetry in both membrane leaflets. The local concentration and, most importantly, its asymmetric distribution in the bilayer are a measure of induced membrane perturbation, which is expected to significantly affect its properties as a cholesterol donor and hydrophobic barrier. In this work we have characterized the partition of several BS, nonconjugated and conjugated with glycine, to large unilamellar vesicles (LUVs) in the liquid-disordered phase and with liquid-ordered/liquid-disordered phase coexistence, using isothermal titration calorimetry (ITC). The partition into the liquid-disordered bilayer was characterized by large partition coefficients and favored by enthalpy, while association with the more ordered membrane was weak and driven only by the hydrophobic effect. The trihydroxy BS partitions less efficiently toward the membranes but shows faster translocation rates, in agreement with a membrane protective effect of those BS. The rate of translocation through the more ordered membrane was faster, indicating accumulation of BS at specific locations in this membrane.

Synthesis and characterization of a lipidic alpha amino acid: solubility and interaction with serum albumin and lipid bilayers.

The lipidic α-amino acid with 11 carbons in the alkyl lateral chain (α-aminotridecanoic acid) was synthesized via multicomponent hydroformylation/Strecker reaction, which is a greener synthetic approach to promote this transformation relative to previously described methods. Its solubility and aggregation behavior in aqueous solutions was characterized, as well as the interaction with lipid bilayers. Lipidic amino acids are very promising molecules in the development of prodrugs with increased bioavailability due to the presence of the two polar functional groups and nonpolar alkyl chain. They are also biocompatible surfactants that may be used in the food and pharmaceutical industry. In this work we have conjugated the lipidic amino acid with a fluorescent polar group (7-nitrobenz-2-oxa-1,3-diazol-4-yl), to mimic drug conjugates, and its association with serum proteins and lipid bilayers was characterized. The results obtained indicate that conjugates of polar molecules with lipidic α-amino acid, via covalent attachment to the amine group, have a relatively high solubility in aqueous solutions due to their negative global charge. They bind to serum albumin with intermediate affinity and show a very high partition coefficient into lipid bilayers in the liquid-disordered state. The attachment of the polar group to the lipidic amino acid increased strongly the aqueous solubility of the amphiphile, although the partition coefficient into lipid membranes was not significantly reduced. Conjugation of polar drugs with lipidic amino acids is therefore an efficient approach to increase their affinity for biomembranes.

Quantification of cholesterol solubilized in bile salt micellar aqueous solutions using (13)C nuclear magnetic resonance.

In this work, we develop a methodology to quantitatively follow the solubilization of cholesterol on glycodeoxycholic acid (GDCA) micelles using (13)C nuclear magnetic resonance (NMR). The amount of solubilized cholesterol enriched in (13)C at position 4, [4-(13)C]cholesterol, was quantified from the area of its resonance, at 44.5 ppm, using the CH(2) groups from GDCA as an internal reference. The loading of the micelles with cholesterol leads to a quantitative upper field shift of most carbons in the nonpolar surface of GDCA, and this was used to follow the solubilization of unlabeled cholesterol. The solubilization followed a pseudo first-order kinetics with a characteristic time constant of 3.6 h, and the maximum solubility of cholesterol in 50 mM total lipid (GDCA + cholesterol) is 3.0 ± 0.1mM, corresponding to a mean occupation number per micelle ≥1. The solubilization profile indicates that the affinity of cholesterol for the GDCA micelles is unaffected by the presence of the solute, leading essentially to full solubilization up to the saturation limit. The relaxation times of GDCA carbons at 50mM give information regarding its aggregation and indicate that GDCA is associated in small micelles (hydrodynamic [Rh] = 1.1 nm) without any evidence for formation of larger secondary micelles. This was confirmed by dynamic light scattering results.