Fatty acid specificity for the esterification of vitamin A and cholesterol by intestinal and pancreatic enzymes in rats

VITAMIN A and cholesterol esters have been shown to undergo extensive hydrolysis in the lumen of the small intestine during the process of absorption; they are re-esterified to appear in the lymph mostly as esters1,2. However, the vitamin A esters of the lymph, blood and liver of the rat are formed by long-chain fatty acids3 and in the normal rat liver, probably as palmitates4. On the other hand, cholesterol esters are usually made up of poly-unsaturated fatty acids in the lymph and blood of rats5. For the absorption of the two lipid materials, the enzymes of the pancreas have been largely implicated, while not much attention has been paid to the possible role of the mucosal enzymes. From the behaviour of the mucosal enzymes, as presented here, it appears that probably these enzymes play a more important part in the re-esterification of the two lipid materials during their absorption.

High cholesterol diet and esterification of cholesterol by the intestinal mucosa of rats

Young male rats maintained on a diet containing 1% cholesterol were sacrificed at the end of 1st, 2nd, 3rd, 5th, and 7th week. Acetone powders prepared from their intestinal mucosa and pancreas were tested for the synthetic and hydrolytic activities for Vitamin A and cholesterol esters. The esterifying activity of the mucosal enzymes for both Vitamin A and cholesterol increased progressively up to the end of the 5th week; the increase in esterification of cholesterol was more marked with respect to saturated fatty acids, as compared to the unsaturated ones. The pancreatic enzymes remained unaffected. It is suggested that one of the reasons for the accumulation of cholesterol esters in animal tissues may be the increased esterification of the sterol in the mucosa induced by dietary cholesterol.

Synthesis, aggregation behavior and cholesterol solubilization studies of 16-epi-pythocholic acid (3 alpha,12 alpha,16 beta-trihydroxy-5 beta-cholan-24-oic acid)

Synthesis, aggregation behavior and in vitro cholesterol solubilization studies of 16-epi-pythocholic acid (3 alpha,12 alpha,16 beta-trihydroxy-5 beta-cholan-24-oic acid, EPCA) are reported. The synthesis of this unnatural epimer of pythocholic acid (3 alpha,12 alpha,16 alpha-trihydroxy-5 beta-cholan-24-oic acid, PCA) involves a series of simple and selective chemical transformations with an overall yield of 21% starting from readily available cholic acid (CA). The critical micellar concentration (CMC) of 16-epi-pythocholate in aqueous media was determined using pyrene as a fluorescent probe. In vitro cholesterol solubilization ability was evaluated using anhydrous cholesterol and results were compared with those of other natural di-and trihydroxy bile acids. These studies showed that 16-epi-pythocholic acid (16 beta-hydroxy-deoxycholic acid) behaves similar to cholic acid (CA) and avicholic acid (3 alpha,7 alpha,16 alpha-trihydroxy-5 beta-cholan-24-oic acid, ACA) in its aggregation behavior and cholesterol dissolution properties. (C) 2010 Elsevier Inc. All rights reserved.

ATP in the regulation of cholesterol biosynthesis- a supra-energetic role

ATP, given intraperitoneally to starved rats stimulates hepatic biosynthesis of sterols at a pre-mevalonate site.

A fluorescent probe study of the solubilisation of cholesterol by biie salt-phospholipid micelles

The fluorescent probe dansyl cadaverine has been shown to bind strongly to mixed bile salt-phospholipid micelles containing unsaturation in the fatty acyl chains. Incorporation of cholesterol into the mixed micelles reduces the number of molecules of bound dansyl cadaverine without altering the binding affinity. These results suggest a tighter packing of the hydrocarbon matrix of the micelles in the presence of cholesterol.

Microsomal redox systems in brown adipose tissue: high lipid peroxidation, low cholesterol biosynthesis and no detectable cytochrome P-450

The presence of redox systems in microsomes of brown adipose tissue (BAT) in cold exposed rats was investigated and compared with liver. BAT microsomes showed high activity of lipid peroxidation measured both by the formation of malondialdehyde (MDA) and by oxygen uptake. NADH and NADPH dependent cytochrome c reductase activity were present in both BAT and liver microsomes. Aminopyrine demethylase and aniline hydroxylase activities, the characteristic detoxification enzymes in liver microsomes could not be detected in BAT microsomes. BAT minces showed very poor incorporation of [1-14C]acetate and [2-14C]-mevalonate in unsaponifiable lipid fraction compared to liver. Biosynthesis of cholesterol and ubiquinone, but not fatty acids, and the activity of 3-hydroxy-3-methyl glutaryl CoA reductase appear to be very low in BAT. Examination of difference spectra showed the presence of only cytochrome b 5 in BAT microsomes. In addition to the inability to detect the enzyme activities dependent on cytochrome P-450, a protein with the characteristic spectrum, molecular size in SDS-PAGE and interaction with antibodies in double diffusion test, also could not be detected in BAT microsomes. The high activity of lipid peroxidation in microsomes, being associated with large oxygen uptake and oxidation of NADPH, will also contribute to the energy dissipation as heat in BAT, considered important in thermogenesis.

Membranes of Cationic Gemini Lipids based on Cholesterol with Hydroxyl Headgroups and their Interactions with DNA and Phospholipid

Two series of cholesterol-based cationic gemini lipids with and without hydroxyl functions at the headgroups possessing different lengths of polymethylene -(CH2)(n)-] (n = 3, 4, 5, 6, 12) spacer have been synthesized. Each gemini lipid formed stable suspension in water. The suspensions of these gemini lipids in water were investigated using transmission electron microscopy, dynamic light scattering, zeta potential measurements and X-ray diffraction to characterize the nature of the individual aggregates formed therein. The aggregation properties of these gemini lipids in water were found to strongly depend upon the length of the spacer and the presence of hydroxyl group at the headgroup region. Lipoplex formation (DNA binding) and the release of the DNA from such lipoplexes were performed to understand the nature of interactions that prevail between these cationic cholesterol aggregates and duplex DNA. The interactions between such gemini lipids and DNA depend both on the presence of OH on the headgroups and the spacer length between the headgroups. Finally, we studied the effect of incorporation of each cationic gemini lipid into dipalmitoyl phosphatidylcholine vesicles using differential scanning calorimetry. The properties of the resulting mixed membranes were found again to depend upon the nature of the headgroup and the spacer chain length.

Syntheses, Transfection Efficacy and Cell Toxicity Properties of Novel Cholesterol-based Gemini Lipids having Hydroxyethyl Head group

We have synthesized five new cholesterol based gemini cationic lipids possessing hydroxyethyl (-CH2CH2OH) function on each head group, which differ in the length of the polymethylene spacer chain. These gemini lipids are important for gene delivery processes as they possess pre-optimized molecular features, e. g., cholesterol backbone, ether linkage and a variable spacer chain between both the headgroups of the gemini lipids. Cationic liposomes were prepared from each of these lipids individually and as a mixture of individual cationic gemini lipid and 1,2-dioleoyl phosphatidylethanolamine (DOPE). Each gemini lipid based formulation induced better transfection activity than that of their monomeric counterpart. One such gemini lipid with a -(CH2)(12)-spacer, HG-12, showed dramatic increase in the mean fluorescence intensity due to the expression of green-fluorescence protein (GFP) in the presence of 10% FBS compared to the conditions where there was no serum. Other gemini lipids retained their gene transfection efficiency without any marked decrease in the presence of serum. The only exception was seen with the gemini with a -(CH2)(3)-spacer, HG-3, which on gene transfection in the presence of 10% FBS lost similar to 70% of its transfection efficiency. Overall the gemini lipid with a -(CH2)(5)-spacer, HG-5, showed the highest transfection activity at N/P (lipid/DNA) ratio of 0.5 and lipid : DOPE molar ratio of 2. Upon comparison of the relevant parameters, e. g., %-transfected cells, the amount of DNA transfected to each cell and %-cell viability all together against Lipofectamine 2000, one of the best commercial transfecting agents, the optimized lipid formulation based on DOPE/HG-5 was found to be comparable. In terms of its ability to induce gene-transfer in the presence of serum and shelf-life DOPE/HG-5 liposome was found to be superior to its commercial counterpart. Confocal imaging analysis confirmed that in the presence of 10% serum using a Lipid : DOPE of 1 : 4 and N/P charge ratio of 0.75 with 1.2 mu g DNA per well, HG-5 is better than Lipofectamine 2000.

Synthesis and vesicle formation from dimeric pseudoglyceryl lipids with (CH2)(m) spacers: pronounced m-value dependence of thermal properties, vesicle fusion, and cholesterol complexation

Eight new dimeric lipids, in which the two Me2N+ ion headgroups are separated by a variable number of polymethylene units [-(CH2)(m)-], have been synthesized. The electron micrograph (TEM) and dynamic light scattering (DLS) of their aqueous dispersions confirmed the formation of vesicular-type aggregates. The vesicle sizes and morphologies were found to depend strongly on the m value, the method, and thermal history of the vesicle preparation. Information on the thermotropic properties of the resulting vesicles was obtained from microcalorimetry and temperature-dependent fluorescence anisotropy measurements. Interestingly, the T-m values for these vesicles revealed a nonlinear dependence on spacer chain length (m value). These vesicles were able to entrap riboflavin. The rates of permeation of the OH- ion under an imposed transmembrane pH gradient were also found to depend significantly on the m value. X-Ray diffraction of the cast films of the lipid dispersions elucidated the nature and the thickness of these membrane organizations, and it was revealed that these lipids organize in three different ways depending on the m value. The EPR spin-probe method with the doxylstearic acids 5NS, 12NS, and 16NS, spin-labeled at various positions of stearic acid, was used to establish, the chain-flexibility gradient and homogeneity of these bilayer assemblies. The apparent fusogenic propensities of these bipolar tetraether lipids were investigated in the presence of Na2SO4 with fluorescence-resonance energy-transfer fusion assay. Small unilamellar vesicles formed from 1 and three representative biscationic lipids were also studied with fluorescence anisotropy and H-1 NMR spectroscopic techniques in the absence and the presence of varying amounts of cholesterol.

2-Halooxyethylene ethers of cholesterol as novel single component, room temperature cholesteric LC materials

A series of 2-haloethoxyethyl cholesteryl ethers has been synthesized. Each material shows attractive liquid-crystalline properties as revealed by differential scanning calorimetry, polarizing microscopy, and temperature-dependence of selective reflection characteristic of the cholesteric mesophase. These are interesting examples of simple, nonpolymeric, single component systems that show the cholesteric mesophase at room temperature.

Advantage of the ether linkage between the positive charge and the cholesteryl skeleton in cholesterol-based amphiphiles as vectors for gene delivery

Twelve novel cationic cholesterol derivatives with different linkage types between the cationic headgroup and the cholesteryl backbone have been developed. These have been tested for their efficacies as gene transfer agents as mixtures with dioleoyl phosphatidylethanolamine (DOPE). A pronounced improvement in transfection efficiency was observed when the cationic center was linked to the steroid backbone using an ether type bond. Among these, cholest-5-en-3b-oxyethane-N, N,N-trimethylammonium bromide (2a) and cholest-5-en-3b-oxyethane-N, N-dimethyl-N-2-hydroxyethylammonium bromide (3d) showed transfection efficiencies considerably greater than commercially available reagents such as Lipofectin or Lipofectamine. To achieve transfection, 3d did not require DOPE. Increasing hydration at the headgroup level for both ester- and ether-linked amphiphiles resulted in progressive loss of transfection efficiency. Transfection efficiency was also greatly reduced when a 'disorder'-inducing chain like an oleyl (cis-9-octadecenyl) segment was added to these cholesteryl amphiphiles. Importantly, the transfection ability of 2a with DOPE in the presence of serum was significantly greater than for a commercially available reagent, Lipofectamine. This suggests that these novel cholesterol-based amphiphiles might prove promising in applications involving liposome-mediated gene transfection. This investigation demonstrates the importance of structural features at the molecular level for the design of cholesterol-based gene delivery reagents that would aid the development of newer, more efficient formulations based on this class of molecules.

Crystal and molecular structure of synthetic cholesterol compounds vitamin D3-analogues (I) 24(S)-hydroxy coprastan-3-one & (II) 24(R)-hydroxy coprastan-3-one

The title compound I (24-(S)-Hydroxy Coprastan-3-one) crystallises in orthorhombic space group P2(1)2(1)2(1) with Z = 4. The unit cell dimensions are a = 6.701(2)Angstrom, b = 11.506(8)Angstrom, c = 32.183(4)Angstrom, V = 2481(2)Angstrom (3), D-cal = 1.077 Mg/m(3). The tide compound II (24-(R)-Hydroxy Coprastan-3-one) crystallises in orthorhombic space group P212121 with two molecules per assymetric unit and with Z = 8. The Unit cell dimensions are a = 10.954(2)Angstrom, b = 21.757(6)Angstrom, c = 21.130(7)Angstrom, V = 5035.0(2)Angstrom (3), D-cal = 1.062 Mg/m(3). In compound I and in both the molecules of compound II, the rings A, B & C are in chair conformation and the five membered ring D is in envelope conformation. The priority sequence attached to the chiral carbon C24 has "S" designation in compound I and "R" designation in compound II. The structures are stabilized by C-H . . .O and O-H---O hydrogen bonds.

Synthesis and characterization of novel cationic lipid and cholesterol-coated gold nanoparticles and their interactions with dipalmitoylphosphatidylcholine membranes

Novel gold nanoparticles bearing cationic single-chain, double-chain, and cholesterol based amphiphilic units have been synthesized. These nanoparticles represent size-stable entities in which various cationic lipids have been immobilized through their thiol group onto the gold nanoparticle core. The resulting colloids have been characterized by UV-vis, (1)H NMR, FT-IR spectroscopy, and transmission electron microscopy. The average size of the resultant nanoparticles could be controlled by the relative bulkiness of the capping agent. Thus, the average diameters of the nanoparticles formed from the cationic single-chain, double-chain, and cholesterol based thiolate-coated materials were 5.9,2.9, and 2.04 nm, respectively. We also examined the interaction of these cationic gold nanoparticles with vesicular membranes generated from dipalmitoylphosphatidylcholine (DPPC) lipid suspensions. Nanoparticle doped DPPC vesicular suspensions displayed a characteristic surface plasmon band in their UV-vis spectra. Inclusion of nanoparticles in vesicular suspensions led to increases in the aggregate diameters, as evidenced from dynamic light scattering. Differential scanning calorimetric examination indicated that incorporation of single-chain, double-chain, and cholesteryl-linked cationic nanoparticles exert variable effects on the DPPC melting transitions. While increased doping of single-chain nanoparticles in DPPC resulted in the phases that melt at higher temperatures, inclusion of an incremental amount of double-chain nanoparticles caused the lowering of the melting temperature of DPPC. On the other hand, the cationic cholesteryl nanoparticle interacted with DPPC in membranes in a manner somewhat analogous to that of cholesterol itself and caused broadening of the DPPC melting transition.