Importance of macrophage cholesterol content on the flux of cholesterol mass

Net flux of cholesterol represents the difference between efflux and influx and can result in net cell-cholesterol accumulation, net cell-cholesterol depletion, or no change in cellular cholesterol content. We measured radiolabeled cell-cholesterol efflux and cell-cholesterol mass using cholesterol-normal and -enriched J774 and elicited mouse peritoneal macrophage cells. Net cell-cholesterol effluxes were observed when cholesterol-enriched J774 cells were incubated with 3.5% apolipoprotein (apo) B depleted human serum, HDL₃, and apo A-I. Net cell-cholesterol influxes were observed when cholesterol-normal J774 cells were incubated with the same acceptors except apo A-I. When incubated with 2.5% individual sera, cholesterol mass efflux in free cholesterol (FC)-enriched J774 cells correlated with the HDL-cholesterol (HDL-C) concentrations (r² = 0.4; P=0.003), whereas cholesterol mass influx in cholesterol-normal J774 cells correlated with the LDL cholesterol (LDL-C) concentrations (r² = 0.6; P<0.0001) of the individual sera. A positive correlation was observed between measurements of [³H]cholesterol efflux and reductions in cholesterol mass (r² = 0.4; P=0.001) in FC-enriched J774 cells. In conclusion, isotopic efflux measurements from cholesterol-normal or cholesterol-enriched cells provide an accurate measurement of relative ability of an acceptor to remove labeled cholesterol under a specific set of experimental conditions, i.e., efflux potential. Moreover, isotopic efflux measurements can reflect changes in cellular cholesterol mass if the donor cells are enriched with cholesterol.     

Effect of cholesterol nucleation-promoting activity on cholesterol solubilization in model bile

Human bile contains a factor with cholesterol nucleation-promoting activity that binds to concanavalin A-Sepharose. In this study we have investigated the effect of this activity on the dynamics of lipid solubilization in supersaturated model bile. A concanavalin A binding protein fraction of human bile was mixed with model bile and the effect on the distribution of cholesterol and phospholipid between mixed micelles and phospholipid/cholesterol vesicles was studied by means of density gradient ultracentrifugation. The nucleation-promoting activity containing fraction induced a transfer of cholesterol and phospholipid from the micellar to the vesicular phase. This led to a decrease in the density of the vesicular fraction. We have also studied the effect of promoting activity on the nucleation time of an isolated vesicle fraction. A decrease of the nucleation time of 10.7 +/- 1.3 to 2.3 +/- 0.3 days was observed. In conclusion, a concanavalin A binding protein fraction from human bile stimulated cholesterol nucleation via a double effect; it increased the amount of vesicular cholesterol and phospholipid, and it also directly induced nucleation of cholesterol from the vesicles.     

Influence of phosphatidylcholine/cholesterol molar ratios in liposomes on cholesterol reactivity with cholesterol:oxygen oxidoreductase]

The reactivity of sonicated phosphatidylcholine-cholesterol liposomes with cholesterol : oxygene oxydoreductase, an enzyme which catalyses the oxidation of the 3 beta hydroxyl group of cholesterol to a ketone group, is compared with that of ternary system phosphatidylcholine-cholesterol-Thesit. Regardless to the phosphatidylcholines nature and the phosphatidylcholine/cholesterol molar ratio (R), the enzymatic oxidation rate of liposomal cholesterol is slower than when the reaction is developed in the present of Thesit, a surfactif agent which destroyes the lamellar particles. This is true whether Thesit is added during preparation of dispersions or during incubation with cholesterol oxydase. The enzymatic oxydation rate of cholesterol of ternary systems phosphatidylcholine-cholesterol-Thesit is independent of the (R) value and the phosphatidylcholine fatty acid unsaturation, whereas that of phosphatidylcholine-cholesterol dispersions depends on these two parameters. The reaction rate increases in the order: dipalmitoylphosphatidylcholine to yolk egg phosphatidylcholines, and dioleylphosphatidylcholine. The optimal conditions for cholesterol oxidation were found to be R = 0.5. This result is not affected by the phosphatidylcholines nature. In order to explain these data, various hypotheses are considered. In particular, the weak liposomal cholesterol reactivity with cholesterol oxidase could result from an inhibitory effect on the enzyme-substrate combination due to the polar phosphorylcholine groups.     

Effects of dietary cholesterol on the regulation of total body cholesterol in man

Studies on the interaction of cholesterol absorption, synthesis, and excretion were carried out in eight patients using sterol balance techniques. Absorption of dietary cholesterol was found to increase with intake; up to 1 g of cholesterol was absorbed in patients fed as much as 3 g per day. In most patients, increased absorption of cholesterol evoked two compensatory mechanisms: (a) increased reexcretion of cholesterol (but not of bile acids), and (b) decrease in total body synthesis. However, the amount of suppression in synthesis was extremely variable from one patient to another; one patient had no decrease in synthesis despite a large increment in absorption of dietary cholesterol, and two patients showed a complete suppression of synthesis. In the majority of cases the accumulation of cholesterol in body pools was small because of adequate compensation by reexcretion plus reduced synthesis, but in a few patients large accumulations occurred on high cholesterol diets when absorption exceeded the compensatory mechanisms. These accumulations were not necessarily reflected in plasma cholesterol levels; these increased only slightly or not at all.     

The effect of digitonin-containing fixatives on the retention of free cholesterol and cholesterol esters

Synopsis The influence of several fixation and dehydration procedures on the retention of free cholesterol and cholesterol esters was studied in filter paper preparations. The retention of free cholesterol by the filter paper proved to be decreased by the addition of digitonin to the aldehyde fixative (aqueous phase) and was only slightly enhanced by partial dehydration (alcoholic phase, up to 70% ethanol). Furthermore, digitonin or the presumably formed cholesterol-digitonide complex bound hardly any osmium oxides in glass-fibre paper.Up to 26% of the cholesterol esters was mobilized during the aqueous phase when digitonin was added to the aldehyde fixative. When the glass-fibre papers containing the digitonin cholesterol-ester-osmate complexes were stored in distilled water after fixation, the fluid became turbid. Particulate material isolated from this turbid solution showed ultrastructurally a close resemblance to the whorls observed by several authors in tissue fixed by a digitonin-containing aldehyde fixative.Digitonin also changed the ultrastructural appearance of liposomes, containing lecithin: cholesterol: phosphatidic acid, in a molar ratio 721. Our observations lead to the conclusion that the use of digitonin-containing fixatives should be abandoned, because they give results which cannot be interpreted. By the use of K₄[Fe(CN)₆] containing OsO₄ in the post-fixation step we were able to demonstrate an increase in the visualization of membranous structures (liposomes).     

Amperometric determination of serum total cholesterol with nanoparticles of cholesterol esterase and cholesterol oxidase

We describe the preparation of glutaraldehyde cross-linked and functionalized cholesterol esterase nanoparticles (ChENPs) and cholesterol oxidase nanoparticles (ChOxNPs) aggregates and their co-immobilization onto Au electrode for improved amperometric determination of serum total cholesterol. Transmission electron microscope (TEM) images of ChENPs and ChOxNPs showed their spherical shape and average size of 35.40 and 56.97 nm, respectively. Scanning electron microscope (SEM) studies of Au electrode confirmed the co-immobilization of enzyme nanoparticles (ENPs). The biosensor exhibited optimal response at pH 5.5 and 40 °C within 5 s when polarized at +0.25 V versus Ag/AgCl. The working/linear range of the biosensor was 10–700 mg/dl for cholesterol. The sensor showed high sensitivity and measured total cholesterol as low as 0.1 mg/dl. The biosensor was evaluated and employed for total cholesterol determination in sera of apparently healthy and diseased persons. The analytical recovery of added cholesterol was 90%, whereas the within-batch and between-batch coefficients of variation (CVs) were less than 2% and less than 3%. There was a good correlation (r = 0.99) between serum cholesterol values as measured by the standard enzymic colorimetric method and the current method. The initial activity of ENPs/working electrode was reduced by 50% during its regular use (200 times) over a period of 60 days when stored dry at 4 °C.