Quantitative Determination of Lipid Classes

Most lipid classes can be quantitated without previous separation on the basis of characteristic constituents or specific properties. If quantitative determination is preceded by preparative procedures specificity and sensitivity of the assays may be increased. Direct quantitative determinations in plasma, without prior extraction of total lipids frequently may yield erroneously high values. 

Cholesterol occurs in most biological materials in both the free and esterified forms. Quantitative determination of cholesterol is based on color reactions, which are not absolutely specific but suffice since cholesterol is the predominating sterol in most materials. The color reactions include the Liebermann-Burchard reaction with sulfuric acid and acetic acid anhydride, the Tschugaeff reaction with zinc chloride and acetyl chloride in acetic acid, or the Lifschiitz reaction with ferric chloride in acetic acid and sulfuric acid. Both free and esterified cholesterol participate in these reactions. Methods for the determination of cholesterol which are commonly used are those of Schoenheimer and Sperry (1934), Sperry and Webb (1950), Zak et al. (1951), Zlatkis et al. (1953). They were recently reviewed by Vanzetti (1964). 

Because of their high sensitivity methods using ferric chloride are recommended. This reagent permits determination of nanogram amounts of cholesterol in microgram quantities of tissues or microliter volumes of serum (Glick et al. 1964). 

Ferrous sulfate may be used instead of ferric chloride (Searcy and Bergqijist 1960). 

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). 

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Methods concerning isolation and quantitative determination of lipid classes occurring in minor amounts, in certain tissues only, and in various pathological conditions are outlined in the appropriate chapters of this book. Further information may be obtained from papers and monographs by Deuel 1951/1957, Lettre, Inhoffen and Tschesche 1954, Hanahan 1960, Fieser and Fieser 1961, Stahl 1962, ZoLLNER and Eberhagen 1965. 

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