Total and Free Cholesterol

Enzymatic methods are commonly used for this measurement, and many of the current methods use a cholesterol esterase to hydrolyze lipoprotein-cholesteryl esters, followed by further reactions where cholesterol oxidase is linked to a peroxidase-chromogen system (Richmond 1992). Eree cholesterol can be measured by omitting cholesterol esterase of the first reaction step, although many reagent formulations prevent this elimination of the esterase. 

Some of the cholesterol esterases used in these analytical systems show varying degrees of specificity toward the different cholesterol esters, and problems have occurred when these reagents are used for several species. For example, in rats, where there is a fivefold-higher concentration of cholesteryl arachidonate ester compared to human plasma, plasma cholesterol may be underestimated with some reagents (Demacker et al. 1983 Noel, Dupras, and Pillion 1983 Wiebe and Bernert 1984 Evans 1986). 

---

Antilipidemic activity. Triglycerides structured lipids from coconut oil, administered to rats at a dose of 10% of diet for 60 days, produced a 15% decrease in total cholesterol and a 23% decrease in LDL cholesterol levels in the serum compared to coconut oil-fed rats. Total and free cholesterol levels in the liver of structured lipid-fed rats were lowered by 31 and 36%, respectively. The triglycerides in the serum and liver were decreased by 14 and 30%, respectively " . Anti-nociceptive activity. Aqueous extract of the husk fiber, administered orally to mice at doses of 200 or 400 mg/kg, produced an inhibition of the acetic acid-induced writhing response . 

T. Yao, M. Sato, Y. Kobayashi, and T. Wasa, Amperometric Assays of Total and Free Cholesterols in Semm by the Combined Use of Immobilized Cholesterol Esterase and Cholesterol Oxidase Reactors and Peroxidase Electrode in a Flow Injection System. Anal. Biochem., 149 (1985) 387. 

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). 

Assays for the determination of cholesterol in routine clinical laboratories include cholesterol esterase and thus quantify total cholesterol (i.e., unesterified and es-terified cholesterol). However, specific assays are also available that lack cholesterol esterase and hence allow the determination of unesterified or free cholesterol. The difference between total and unesterified cholesterol gives the concentration of cholesterol esters. 

Duncan, I.W., Culbreth, P.H., and Bartis, C.A. 1979. Determination of free, total and esterified cholesterol by high-performance liquid chromatography. J. Chromatogr. 162 281-292. 

Sierksma, A., Weststrate, J.A., and Meijer, G.W., Spreads enriched with plant sterols, either esterified 4,4-dimethylsterols or free 4-desmethylsterols, and plasma total- and LDL-cholesterol concentrations, Br. J. Nutr., 82, 273, 1999. 

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. 

In both obstructive jaundice and parenchymal liver disease the amount of plasma free cholesterol is increased and the percentage of phosphatidylcholines among the total phospholipids is raised. Studies [159] have characterised the different hpopro-teins that occur in both parenchymal liver disease and obstructive jaundice and have related many, but not all, of the changes to reduced plasma LCAT activity. Three separate LDL particles are present in obstructive jaundice (lipoprotein-X (LP-X), large particles rich in triglyceride and free cholesterol, and normal-sized particles with less cholesterol ester and more triglyceride than usual. In parenchymal liver disease only the normal-sized particle is found, although occasionally LP-X may occur. Abnormalities have been demonstrated in HDL and VLDL in patients with both types of liver disease when LCAT activity was low [160]. 

Cholesterol esters are considerably more hydrophobic than cholesterol itself. The amounts of cholesterol and cholesterol esters associated in blood lipoprotein complexes called LDL are typically about two-thirds of the total plasma cholesterol (total plasma cholesterol ranges from 130 to 260 mg/100 mL of human plasma, with the most desirable levels between 160 and 200). More than 40% of the weight of the LDL particle is cholesterol esters, and the total of esterified and free cholesterol amounts to well over half the total weight. 

Liver Cholesterol. Liver TC and free cholesterol (FC) content were measured according to Carlson and Goldfarb (23). Esterified cholesterol (EC) was calculated by the difference between total cholesterol and free cholesterol. 

Determination at Two Temperatures, of Total and Free Plasma Cholesterol by Gas-Liquid Chromatography. Proposal of a Reference Method Pharm. Biol. 11(108) 139-142 (1977) CA 87 49571x... 

Total Cholesterol and Free Cholesterol by Gas-Liquid Chromatography. Suggestion for a Reference Method Clin. Chim. Acta 68(1) 31-41 (1976) CA 84 176068b... 

Sierksma et al. (1999) reported that plasma total and LDL cholesterol levels were 3.8% and 6% lower, respectively, in those consuming a spread containing 0.8 g free sterols daily than in those consuming a control spread. Volpe et al. (2001) incorporated soy sterols into a low-fat yoghurt-based drink. With 1 g of sterols, semm total and LDL cholesterol levels were reduced (but apparently not significantly) by 4.4% and 6.2%, respectively, compared with a control. In an uncontrolled follow-up study with 2 g of sterols, serum total and LDL cholesterol levels decreased by 11.2% and 15.6%, respectively, from baseline levels. Tikkanen et al. (2001) recently studied the effects of free sterols incorporated into various low-fat food products, such as yoghurt, meat, and jam. Serum LDL cholesterol levels were reduced by 8% compared with a control group when sterol doses of up to 5 g/day were used. 

Diabetic people are more likely to have dyslipide-mia than nondiabetic people. When control of diabetes is lost, patients may demonstrate gross hypertriglyceridemia due to increased production of very-low-density lipoprotein (VLDL) particles in the liver as a consequence of the increased flux of free fatty acids from the peripheral tissues. At the same time total and LDL cholesterol may be raised. Improvement in diabetic control often achieves normalization of blood lipids, but where hyperlipidemia persists there may be a place for use of dietary fiber, especially soluble fiber, and especially oat yS-glucan-containing foods as an adjunct to dietary and pharmacological therapy (see above). 

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. 

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. 

ALTERNATE PROTOCOL 2 ENZYMATIC MEASUREMENT OF CHOLESTEROL Test combination kits for enzymatic determination of cholesterol in food are now commercially available. For the determination of total cholesterol, esterified cholesterol is hydrolyzed to free cholesterol and fatty acid under mild alkaline conditions. Cholesterol oxidase oxidizes free cholesterol to cholest-4-en-3-one to generate hydrogen peroxide, which further oxidizes methanol to formaldehyde. Formaldehyde then reacts with acetyl acetone in the presence of NH4+ ions to form yellow lutidine dye, which is subsequently determined spectrophotometric al 1 y. 

---