Cholesterol ester analysis

 CHOLESTEROL  ESTER

ANALYSIS

Cholesterol esters are largely spread in body fluids of animals (plasma lipoproteins) and may be also found in vessel walls as fatty streaks in atherosclerosis. Acylated sterols are also found in plant structures, their analysis is similar to that of cholesterol esters.

The amount of cholesterol esters may be determined after TLC purification either by cholesterol or fatty acid analysis. In the later case, a known amount of an internal standard (C17:0) is added before further treatment.

The separation of free and esterified sterols may be also effected by column chromatography (SPE).
Oil samples are transferred by aid of hexane onto a silicagel column. Elution is started with hexane/ethyl acetate (90/10, v/v) to collect the steryl esters, followed by elution with hexane/ether/ethanol (25/25/50, v/v) for collection of free sterols. After evaporation of the solvent, both fractions can be derivatized according to the selected procedure (after saponification for the steryl ester fraction) (Verleyen T et al., JAOCS 2002, 79, 117).

The fatty acid composition of cholesterol esters is determined by gas chromatography after either saponification or direct transmethylation. During hydrolysis and methylation, contaminating residues are formed and were shown to interfere with the GC analysis (Smuts CM et al, J Chromatogr 1991, 564, 272). It must be noticed that this interference is predominant in the retention zone of the polyunsaturated fatty acids.
A simple mean to eliminate this problem is to chromatograph the fatty acid methyl esters by TLC (normal silica gel plates) in hexane/ether/acetic acid (70/30/1, v/v). Areas containing the methyl esters are visualized by primuline spray, scraped into glass-stoppered vials, suspended in 2 ml methanol and extracted after addition of 1 ml water and 2 ml hexane. The hexane fraction is evaporated and submitted to GC analysis.

An improvement in the preparation of fatty acid methyl esters from sterol esters was described using an alkali-catalyzed methanolysis (Ichihara K et al., JAOCS 2003, 80, 933). Sterol esters were methanolyzed in 0.08 ml methyl propionate with 0.12 ml 0.84 M NaOH in methanol dried with Molecular Sieves 3A. The reaction was terminated by addition of 0.01 ml acetic acid, 0.1 ml hexane containing methyl heptadecanoate as an internal standard, and 0.2 ml water. After vortexing, the hexane layer was injected directly in a GC column. According to that procedure, a yield more than 90% was reached after a 1 h reaction at 37°C. It was shown than a lower yield (about 87%) was reached when cholesterol esters were adsorbed by the silica gel of the TLC plate. Elution of esters from the silica matrix is therefore recommended before composition analysis.

As determination of cholesterol esters in cells and fluid is of great importance in fundamental and clinical research, several methods of measurement using enzyme systems were described. In most of the described procedures, the cholesterol ester amount is calculated in substracting the free cholesterol amount from the total cholesterol amount. Several commercial kits using that approach may be found on the market.
A more precise procedure based on the direct measurement of cholesterol esters has been described (Mizoguchi T et al., J Lipid Res 2004, 45, 396). In the first step, the free cholesterol is oxidized by cholesterol oxidase producing hydrogen peroxide which is decomposed by catalase into water and oxygen. At the second step, cholesterol esters are measured by enzymatic determination (cholesterol esterase and oxidase) and finally by colorimetry or fluorimetry. That method has a very good reproducibility and a high sensitivity.

Other fatty acid esters of steroids

These compounds mainly circulate in the blood but their biological role is poorly known. An efficient analytical method for simultaneous determination of 12 esters in serum has been described (Jung HJ et al., J Chromatogr A 2009, 1216, 1463). The method involves solid-phase extraction to isolate of esters from interfering species, especially cholesteryl esters, and conversion to trimethylsilyl ether derivatives for the direct analysis by gas chromatography–mass spectrometry.