Cholesterol

CHOLESTEROL, C₂₇H₄₆O, a substance from the sterol group, is a zoosterin, as it occurs exclusively in the animal organism; it is found in the composition of apparently all tissues and organs and almost all body fluids either in free form or in the form of esters with higher fatty acids: stearic, palmitic, and oleic. It is particularly abundant in nervous tissue, brain tissue, skin fat, kidneys, bile; human gallstones consist almost entirely of Ch. For information on the content of Ch. in blood, see Blood (however, the figures for Ch. content cannot be considered accurate, as they vary greatly among different authors depending on the use of various determination methods and non-observance of identical conditions). Ch. is also usually found in the form of crystals in old transudates, in the contents of cysts, in atheromas, in pus, in old foci of fat infiltration; then in egg yolk, semen, meconium, excrements, sometimes in urinary stones. Esters of Ch., especially in large quantities, are found in the fat of sheep's wool (together with esters of the so-called isocholesterol), in blood, in the lymph of mammals and birds, in the fluid in chylous ascites, in tuberculous nodules, in vernix caseosa, in hair, feathers, hooves, in the epidermis in dermatitis exfoliativa, in the large white kidney and other parenchymatously altered organs. In general, it should be considered, in contrast to old data, that esters of Ch. are found in the same places as free Ch., and in fluids the content of esters of Ch. is 2-3 times greater than that of free Ch. Ch. is close to terpenes and represents a hydroaromatic monatomic secondary alcohol with one unsaturated bond. As a result of the work of Rosenheim and King, Wieland and Dane, Bernal, Windaus, Bonstedt, the structure of cholesterol can probably be expressed by the following formula: CH₃ CH₃

CH₃ I

/

\ CH_a C

CH₂

CH₃ CH₃| C | D | CH_g| CH CH-CH₂ (Bonstedt, 1933). CH_a C

Cholesterol is usually obtained from gallstones. Pure cholesterol is a white, pearly crystalline mass, somewhat fatty in appearance; when left in air and light, X. turns yellow. X. is insoluble in water, as well as in diluted acids and even in strong alkalis, which do not change it even when boiled; it is almost insoluble in cold alcohol, dissolves in 9 parts of boiling alcohol with specific gravity 0.84, in 5.5 parts of boiling alcohol with specific gravity 0.82; it dissolves easily in benzene, ether, chloroform, and other organic solvents; its easy solubility in ethyl acetate allows for the separation of X. from other lipoids; X. also dissolves in oils, in solutions of bile acids, better with the addition of soap; it dissolves poorly in aqueous soap solutions. From anhydrous alcoholic solutions, X. crystallizes in the form of needle-shaped crystals containing no water; from aqueous alcohol and ether, characteristic crystals are formed in the form of colorless transparent rhomboidal tablets, often grown together in steps: angles 76°30' or 87°30'; they contain one molecule of crystallization water; when left in air, they lose their water. When dried in a vacuum, X. has a melting point of 148.5°. Cholesterol rotates the plane of polarization of light to the left: [α]° = -31.12 (for a 2% ethereal solution of anhydrous cholesterol). X. gives a series of color reactions which can be used for its detection. 1. Salkowski's reaction: when a solution of X. in chloroform is mixed with an equal volume of concentrated H₂SO₄, the X. solution first turns red, then passing to violet-red, while the lower layer (sulfuric acid) turns yellow-red and shows a green fluorescence. 2. Lieberman-Burchard reaction. To a solution of X. in 2 cm³ of chloroform, 10 drops of acetic anhydride and drops of concentrated H₂SO₄ are added. The mixture first turns pink-red, then violet, blue, passing to green. Some methods for the quantitative determination of X. are based on the ability of X. to form difficultly soluble compounds with saponins, in particular with digitonin. The physiological role of X. appears in a new light as a result of recent works which have established a direct genetic connection of X., on the one hand, with bile acids, on the other hand, with sex hormones: folliculin, the hormone of the corpus luteum, and the male sex hormone androsterol. Substances related in structure to X. are also found in substances causing experimental cancer (so-called carcinogenic substances): they, like X., are derivatives of polycyclic hydrocarbons chrysene and phenanthrene (see Estrogenic substances). Being a hydrophobic colloid, together with other lipoids it participates in the regulation of osmotic and diffusion phenomena in the cell, being part of its shell; in the hemolysis of erythrocytes by cobra venom and others, it has an inhibitory effect, opposite to lecithin; esters of X. do not possess this property. And in other actions affecting the physicochemical processes of the organism, X. is to a certain extent an antagonist of lecithin. The esters of X., which are part of sebum, apparently play a protective role for the skin, similar to wax in plants. Little is yet known regarding the origin and transformations of X. in the organism. Its source in the organism is first of all the X. of food. How the breakdown of esters of X. and its absorption in the intestine proceeds is not clarified; it is assumed that part of the esters undergoes breakdown, part is absorbed unchanged; for the absorption of X. the presence of bile acids and saponified fats is necessary. In addition, the organism is capable of synthesizing X., but from what substances is unknown. There are assumptions that the material for the synthesis of X., as well as for the synthesis of bile acids, are unsaturated hydrocarbons: squalene, C₃₀H₅₀, spinacene, C₂₉H₄₈, first found in the liver of sharks (Tsujimoto, Chapman), and then also found in the fat of dermoid cysts of man; this would establish a genetic connection between X. and bile acids, which are so close to each other in structure. Part of X. is excreted together with bile into the intestine, where it partially, under the action of microorganisms, turns into coprosterol—a product of reduction and isomerization of X.; partly X. is excreted directly through the intestinal wall, as well as through the sebaceous glands. With urine, only insignificant traces of X. are normally excreted (0.25 mg per day); greater excretion indicates a pathological condition of the kidneys; in chyluria, the content of X. can reach 2.4-3.8%. Along with X. and its esters, isocholesterol and its esters are found in the organism. According to new data, isocholesterol is a heterogeneous substance and consists of agnosterol, C₂₀H₄₈O, and lanosterol, C₃₀H₅₀(X?), substances not directly related to X. It gives the Lieberman-Burchard reaction; it gives the Salkowski reaction very weakly. Method for the quantitative determination of X. in blood by Autenrieth. The method is based on the color Lieberman-Burchard reaction. Reagents: 1) 25% solution of KOH, 2) chloroform (anhydrous), 3) anhydrous Na₂SO₄, 4) acetic anhydride, 5) strong H₂SO₄ (chemically pure), 6) standard solution of X.: a) 200 mg X. are dissolved in 100 cm³ of chloroform; b) before the experiment, 1 cm³ of this solution is diluted in 25 cm³ of chloroform. Determination: to 1 cm³ of serum in a wide test tube or in a small flask, add 10 cm³ of alkali, shake well and place for two hours in a boiling water bath. After cooling, the mixture is poured into a separatory funnel; the test tube is rinsed with 5 cm³ of chloroform, which is also poured into the funnel. After the separation of the two layers, the chloroform is drained through a stopcock into a dry flask. The extraction of the aqueous layer remaining in the funnel with chloroform is repeated 3 times, taking each time 5 cm³ of chloroform. The solution of X. in chloroform is dehydrated by shaking with 5 g of anhydrous Na₂SO₄. Then the liquid is filtered into a measuring flask, the vessel and filter are washed with 5 cm³ of chloroform and the chloroform is made up to 25 cm³. 5 cm³ of the obtained extract correspond to 0.2 cm³ of blood. Color reaction: to 50 parts of chloroform extract and simultaneously to 50 parts of standard solution, add 20 parts of acetic anhydride and 1 part of concentrated H₂SO₄, shake and place in a dark thermostat at a temperature of 32-35° for 15 minutes, after which it is colorimetrically measured: if Autenrieth's colorimeter is used, comparison is made according to a special wedge. Calculation: since the concentration of X. in the standard = 8 mg per 100 cm³, then the concentration of X. in the extract = (H_e/H_s) × 8 mg in 100 cm³ of extract (H_e = height of the extract column, H_s = height of the standard solution column); since 100 cm³ of extract equals 4 cm³ of serum, multiplying this result by 25, we obtain the amount of X. in mg%. -Quantitative determination of X. in urine by Windaus. Principle: X. is precipitated by digitonin in the form of digitonin-cholesterol, C₃₆H₆₀O₆; the obtained compound is weighed. Execution: the residue obtained by evaporation of the ether extract of lipoids is boiled with 3 times its volume of 95% alcohol; the solution is decanted and to it is added a 1% solution of digitonin in hot 95% alcohol until precipitation ceases. After several hours, the precipitate is filtered through a Gooch crucible, first washed with 95% alcohol, then with ether. The crucible with the precipitate is dried at 100° to constant weight and weighed. Calculation: multiplying the obtained weight of digitonin-cholesterol by 0.2431, the corresponding amount of X. is found.