Cholesterol, solution preparation

P-Formyl-B-nor-5 -cholestane-3, 5P-diol 3-Acetate (74). To an ozonized solution of 5 g of cholesterol acetate prepared as above is added 10 g of zinc dust followed by dropwise addition of 40 ml of acetic acid with stirring and... 

For each cholesterol standard concentration, prepare a mixture of 1 ml working 5a-cholestane solution (internal standard) and 1 ml working cholesterol solution (standard) in a 5-ml screw-cap glass vial. 

Underestimation of cholesterol Stabilizer in commercially available methanolic KOH solution may inhibit cholesterol oxidase Prepare fresh solution in the laboratory... 

PROBLEM 11.6 What is the molality of a solution prepared by dissolving 0.385 g of cholesterol, C27H460, in 40.0 g of chloroform, CHC13 What is the mole fraction of cholesterol in the solution ... 

Set up the reactions described in the table below in 1.5-ml microcentrifuge tubes The extracted lipids that are currently dissolved in chloroform cannot be used directly in this assay, since the chloroform will denature the enzymes that will eventually produce a colored product (see Fig. 13-1). To prepare the lipids in your sample for this assay, add the indicated volume of lipids in CHCf to tubes 4 and 5, dry them under a stream of N2, and resuspend each sample in 10 pi of isopropanol. The standard cholesterol solutions (tubes 1-3) can be used directly in this assay by adding the indicated volumes to the appropriate tubes. (See chart below.)... 

Semm cholesterol exists as a mixture of fatty acid esters and free cholesterol. Quantitation of total cholesterol involves the initial conversion of the esters to free cholesterol, followed by the total conversion of free cholesterol to its oxidation product. This reaction is coupled to the familiar dye-peroxidase indicator reaction. The parameter A50o measurements using stock cholesterol solutions provide a calibration curve. A reagent blank solution is prepared using all components except cholesterol, and this value is subtracted from all measured A50o values, correcting for any background oxidation of the dye. 

A sample of synthetic cholesterol was prepared consisting entirely of (+)-cholesterol. This synthetic (+)-cholesterol was mixed with some natural (-)-cholesterol. The mixture had a specific rotation of -13°. What fraction of the mixture was (+)-cholesterol Note You need to use the solution to Problem 7.6 for the specific rotation of (-)-cholesterol.)... 

A series of standard solutions of cholesterol were prepared by serial dilution. Equal volumes (2 pi) of a solution of known concentration of the sample and of each of the standard solutions were spotted on to the adsorbent alternatively. For high accuracy a micrometer syringe should be used, but also, a capillary pipette (Drummond Microcap), could be used. 

Cholestenone. Place a mixture of 1 0 g. of purified cholesterol and 0-2 g. of cupric oxide in a test-tube clamped securely at the top, add a fragment of Dry Ice in order to displace the air by carbon dioxide, and insert a plug of cotton wool in the mouth of the tube. Heat in a metal bath at 300-315° for 15 minutes and allow to cool rotate the test-tube occasionally in order to spread the melt on the sides. Warm with a few ml. of benzene and pour the black suspension directly into the top of a previously prepared chromatographic column (1) rinse the test-tube with a little more benzene and pour the rinsings into the column. With the aid of shght suction (> 3-4 cm. of mercury), draw the solution into the alumina column stir the top 0 -5 cm. or so with a stout copper wire to... 

Prepared by refluxing a solution of 5 g. of cholesterol in 7.5 cc. of acetic anhydride for one hour, cooling, and washing the crystalline product with cold methanol yield 5 g., m.p. 114-115 . 

A solution of hydrazoic acid (prepared from about 30 g sodium azide) in ca. 200 ml chloroform is prepared in a well-ventilated hood. Cholesterol (15 g) is dissolved in the hydrazoic acid solution and 3.5 ml of triethylamine is added. The reaction mixture is then stirred at room temperature while 7 g of A-chlorosuccinimide is added. The reaction mixture is allowed to stand overnight and then the chloroform solution is washed successively with dilute sodium bisulfite, dilute soldium bicarbonate solutions and finally with water. The chloroform extract is then dried (Na2S04) and the solvent removed in vacuo. The residue is crystallized from ethanol to yield ca. 8.5 g of (101) in colorless needles mp 138-139°. The chloro azide is reduced to the aziridine by lithium aluminum hydride according to the foregoing procedure. 

When liposomes are prepared from a molecular mixture of lipid components it is important that all lipids be homogeneously dissolved in an organic solvent in order to obteiin bilayers with evenly distributed lipids after hydration. For example, the solubilities of phosphatidylcholine and cholesterol in chloroform are similar their solubility in benzene differs. Upon removal of benzene from the lipid solution an inhomogeneous lipid film is formed on the glass wall and... 

An example of a lipid mixture preparation based on mass would be to dissolve 100 mg of PC, 40 mg of cholesterol, and 10 mg of PG in 5 ml of chloroform/methanol solution. When using activated PE components, inclusion of 10 mg of the PE derivative to this recipe will result in a stable liposome preparation. 

Prepare a biotinylated liposome construct by first dissolving in chloroform, the lipids DMPC cholesterol dicetylphosphate (Sigma) at mole ratios of 5 4 1, and adding to this solution 0.1 mol percent B-PE. Larger mole ratios of B-PE to total lipid may result in nonspecific aggregation of liposomes in the presence of avidin. Maintain all lipids under an inert atmosphere to prevent oxidation. 

Cationic lipids cannot be dissolved in water and form aggregates in aqueous solution, such as bilayers. To prepare a homogeneous reagent, in most cases liposomes were made from cationic lipids in a first step. When it is not possible to form stable lipid bilayers (i.e., liposomes) using a single lipid, then it may be necessary to combine the cationic lipid with one or more so-called helper lipids like cholesterol (Choi) (41) or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) (42). 

In a like manner, a co-polymer of styrene and acryloxysuccinimide with a 10 to 1 ratio was prepared. Enzymes immobilized on this type of polymer had different physical properties. They are soluble in organic solvents such as dioxane and DMF, but insoluble in aqueous solutions. Lipases and cholesterol esterase immobilized on this type of polymer are very stable and active in several organic solvents, and have been used in several enantioselective transformations. The protocols for the immobilization are depicted in Figure 13. 

An instrument was proposed for measurement of Li concentration in human serum, based on the turbidimetric or nephelometric determination of the maximum rate of precipitation of a Li complex, which is related to the Li concentration in the serum. The precipitating reagent was prepared by dissolving KIO4 and FeCls in a KOH solution. Measurements were made every 10 s for 5 min at 550 nm. No interference was recorded for the presence of triglycerides, cholesterol, Na or... 

To prepare proteoliposomes, 7 mg egg yolk phosphatidylcholine, 1.16 pg cholesterol, 77.5 pg cholesteryl oleate and 10 pCi [3H]-cholesteryl oleate, all dissolved in chloroform are mixed. After evaporation of chloroform with nitrogen, the lipids are resolved in 400 pi ethanol. The ethanolic solution is injected into 5 ml of a vortex-ing buffer with 39mmol/l sodium phosphate, 0.01% EDTA, 2 mmol/1 NaN3, and 12 mmol/1 sodium cholate (pH 7.4) 3 mg apoA-I is added. The solution is subsequently dialyzed against a buffer with 39 mmol/1 sodium phosphate, 0.01 % EDTA, 2 mmol/1 NaN3, and 12 mmol/1 sodium cholate (pH 7.4) at 4°C. At the end the solution is filled up with analysis buffer containing 39 mmol/1 sodium phosphate,... 

Prepare a sample blank (without cholesterol oxidase) by pipetting 5 ml catalase/ace-tylacetone/methanol solution and 0.4 ml sample solution into a 10-ml glass test tube. 

Prepare a 1.0 mg/ml cholesterol (>99% pure) stock standard solution in dimethylformamide (DMF). Store up to 1 week at 4°C. Prepare working solutions by diluting the stock solution with DMF to obtain concentrations ranging between 0.05 and 0.5 mg/ml. Prepare fresh. 

The potassium hydroxide solution should be prepared fresh before use, because commercially available methanolic potassium hydroxide solution usually contains a stabilizer that may inhibit cholesterol oxidase. The amount of cholesterol present in the test tube should be between 8 and 160 Ag under the presented conditions to measure a sufficient difference in absorbance. The sample solution should be diluted as shown in Table D 1.3.1. 

A. Cholesterol dibromide. In a 4-1. beaker 150 g. (0.39 mole) of commercial cholesterol (Note 1) is dissolved in 1 1. of absolute ether by warming on the steam bath and stirring with a stout glass rod the solution is then cooled to 25°. A second solution is prepared by adding 5 g. of powdered anhydrous sodium acetate (0.06 mole) (Note 2) to 600 ml. of acetic acid, stirring the mixture and breaking up the lumps with a flat stirring rod 68 g. (0.4 mole) of bromine is then added, and the solution is poured with... 

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