Chemical determinations of cholesterol have been performed in both clinical and research laboratories for many decades. The classical, colorimetric Leiberman-Burchard reaction, described before the turn of the century, has spawned many modifications in efforts to overcome three major problems: I) the presence of bile pigments and non-cholesterol sterols which contribute to false-high values; 2) the differing color intensities developed by free and esterified cholesterol, and 3) inadequate sensitivity. (Detailed reviews and critical discussions have been written.~'2 The modification of Leiberman-Burchard called the "FeCI 3 Method,"3 and the reaction of cholesterol with the aromatic aldehyde, o-phthalaldehyde, 4'5 which is based upon the Kamarowsky reaction, 6'7 remain useful and economical chemical methods. Since the advent of enzymatic methods, 8~1 the use of chemical procedures has declined rapidly; however, it is important to note that commercial secondary calibrators for auto-analyzers in use today often are standardized against these older chemical methods, as are new methods arising from specific research considerations.~Β°~4 The ease and sensitivity of the enzyme assay justifies its wide acceptance for most applications.
Historically, improvements in methodology to quantitate cholesterol were aimed at reaching a better understanding of cholesterol metabolism in blood, while more recently this technology has been applied to the study of other fluids, such as bile, and various tissues from different animal species. Extraction of hydrolyzed tissue into organic solvents ~5 has aided this quest. The need to distinguish unesterified cholesterol (UC) from esterified cholesterol (EC) and to identify the fatty acyl chains of these esters prompted two developments. One method relies upon the precipitation of UC as its digitonide; total cholesterol (TC) is measured similarly following alkaline hydrolysis, 16'j7 and EC is calculated from the difference (TC-UC). The other more recent method separates the major neutral lipid classes by silica chromatography, usually high performance liquid chromatography (HPLC), or thin layer chromatography (TLC). The latter approach will be described below. When interfering compounds are present, such as very high triglyceride (TG) levels or other sterols which are reactants in both the chemical and enzymatic assays, gas liquid chromatography (GLC) becomes the method of choice. ~8
This paper focuses on basic manual methods appropriate for the wide variety of samples generated within research laboratories that can easily be handled by skilled technicians with emphasis on accuracy as the most important feature.