Cholesterol digitonin precipitation

The chemical methods for the quantitation of cholesterol measure total cholesterol, i.e. free and esterified, and so a digitonin precipitate must be prepared if free cholesterol is to be measured. Enzymic methods do not measure the esters and a hydrolysis stage, either chemical or enzymic (using cholesterol ester hydrolase, EC 3.1.1.13), is necessary for the measurement of total cholesterol. 

GC assays of vitamin E have always suffered from the interference of cholesterol. A separation from a-tocopherol proved feasible on capillary columns and rarely on packed columns but at the expense of long analysis times. More commonly, cholesterol was exhaustively removed from saponified extracts using digitonin precipitation, open column chromatography on fiorisil, alumina or cel-ite-digitonin and TLC. Only capillary GC allowed to determine tocopherols (as their TMS ethers) in plasma using the simple solvent extraction approaches typical of LC assays. 

Wool Alcohols. Consists of the free alcohols obtained by saponification of wool fat and contains cholesterol, lanosterol and higher fatty alcohols. It is required by the B,P, to contain not less than 28 0 per cent of cholesterol by digitonin precipitation this is a general method of determination of true sterols. 

In many tissues cholesterol and other sterols exist as a mixture of the free alchohol and its long chain fatty acid ester (esterified at position 3 of the steroid nucleus). The determination of the cholesterol content of a sample may involve the measurement of either of these two fractions individually or the total cholesterol. It is possible to precipitate free cholesterol by adding an equal volume of digitonin (1 gl-1 in 95% ethanol), a naturally occurring glu-coside, to form a complex that is insoluble in most solvents, including water. 

Free cholesterol is determined after precipitation by digitonin. The difference between total and free cholesterol corresponds to the esterified fraction. For estimation of cholesterol esters the value for ester cholesterol is multiplied by the factor 1.67 which is derived from the mean molecular weight of cholesterol ester fatty acids (Bragdon et al. 1956). 

Dissolve about 0 1 g in 12 ml of 95 per cent ethanol and allow to stand overnight for precipitation of some of the fatty alcohols which would otherwise precipitate with the digitonide. Filter and wash with a minimum of ethanol. To the filtrate and washings add 40 ml of hot 0 5 per cent solution of digitonin in 95 per cent ethanol. Collect the precipitate in a Gooch crucible, wash with ethanol and dry at 105°. 1 g digitonide = 0-239 g cholesterol. 

Cholesterol.—Cholesterol forms a characteristic precipitate with the alkaloid digitonin, by means of which it may be isolated and estimated. It also gives a great variety of colour reactions, notably —... 

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