Structure and nomenclature
Determination of free sterols
Determination of cholesterol esters
Determination of sterol glycosides
Determination of acylated steryl glycosides
Cholesterol is the main sterol present in animal tissues but other sterols may be present in biological extracts as those prepared from vegetal.
In animal tissues, the lipidologist is most frequently faced with the determination of cholesterol in complex lipid matrices.
The choice of a strategy
A. If determinations of other
components must be made on the same extract (phospholipids, triacylglycerols..) or if only
some little amount of material is available, it is most convenient to separate cholesterol
(and sterols) by TLC before its quantification.
After chromatography (TLC), sterols (free and esterified) can be detected and positively identified by means of various spray reagents, the following being a popular and sensitive one.
Procedure: the developed TLC plate is sprayed with a solution freshly prepared of 50 mg ferric chloride (FeCl3) in a mixture of 90 ml water, 5 ml acetic acid and 5 ml sulfuric acid. After heating at 100°C for 3-5 min, the sterol spots are indicated by a red-violet color.
When the cholesterol is detected on one part of the TLC (standard lane), the sample spots are scraped and eluted two times with 2 ml dichloromethane. The solvent is evaporated and the residue is dissolved in a know little amount of the same solvent before the determination of cholesterol by any of the proposed methods.
B. If sterols are the only compounds to be determined in the lipid sample, or if the amount of lipids allows several samplings, it is recommended to do a saponification in alkaline conditions before quantification of sterols. Note that this step converts sterol esters into free sterols. To estimate the free and esterified sterols, see below.
small glass tubes, vortex, heated bath (60°C)
3% KOH in methanol
An aliquot of plasma (50-100 µl) or lipid extract is saponified by adding 1 ml of KOH solution and heating 15 min at 60°C.
After cooling, add 1 ml water and extract cholesterol by mixing two times with 2 ml hexane. The hexane phase is evaporated and the unsaponifiable fraction is dissolved in 1 ml of dichloromethane.
After these separation and purification steps the lipidologist must choose its quantification approach :
1. In lipid extracts from animals, cholesterol is generally the only sterol to be determined.
If the amount of cholesterol present in a convenient aliquot is estimated to be in the range 1-10 µg, a colorimetric method can be run.
2. If the cholesterol amount is too low to be estimated by colorimetry (range 0.01-1 µg in the aliquot) or if other sterols are present (food or plant extracts) or if all sterol compounds must be quantified, two techniques can be used:
Quantification by HPLC
Quantification by GLC
C. For the determination of cholesterol in biological
solutions or lipid extracts, a simple and sensitive colorimetric method is presented
below. As other chemical methods, it has no specificity for one sterol or another, but is
very useful for the determination of cholesterol in µg amounts in cellular or plasma
lipid extracts. Reliable results are obtained after removing the other lipids (fatty
acids) by saponification of the extract before cholesterol determination.
Small glass tubes, vortex, spectrophotometer
Dichloromethane - o-phthalaldehyde (OPA) - acetic acid - sulfuric acid - cholesterol.
One aliquot of the dichloromethane solution (100-200 µl) is evaporated, then, add 1 ml of a fresh solution of 50 mg OPA in 100 ml pure acetic acid, vortex and after 10 min (in vortexing) add 0.5 ml concentrated sulfuric acid. After 15 min, the absorbance of the unknown and a series of standards and one blank are determined at 550 nm.
1 to 5 µg of cholesterol can be accurately determined, we use this method for up to 30 µg cholesterol despite a non-linear dose/absorbance relationship. A linear equation is used from 0 to 10 µg and a polynomial equation from 10 to 30 µg.
OTHER ANALYTICAL METHODS
- Gas chromatographic determination of major sterols in edible oils and fats using solid-phase extraction in sample preparation
Toivo J et al., Chromatographia 1998, 48, 745
of cholesterol in foods using non-aqueous capillary electrophoresis
Xu X et al., J chromatogr B 2002, 768, 369
Lipids of food samples are extracted and saponified. Cholesterol is extracted with hexane. The lipid residue is dissolved in the running buffer (100 mM Na acetate in methanol / 100 mM acetic acid in methanol, 19/1). The electrophoresis is carried out at 23.5 kV, 25°C with a fused-silica capillary (50 mm ID x 47 cm) and a Beckman P/ACE 5510 system, the detection is at 210 nm.
The cholesterol retention time is 15 min and the detection limit is 5 mg/ml.
- Analysis of free and esterified
sterols in vegetal oils
Verleyen T et al., JAOCS 2002, 79, 117
- A simplified method for the
quantification of total cholesterol in lipids using gas chromatography
Hwang BS et al., J Food Comp Anal 2003, 16, 169
100 mg of lipid are saponified, followed by methylation with boron trifluoride. The mixture is extracted with ether, added with an internal standard (5a-cholestane) and analyzed by gas chromatography.
- Solid-phase extraction-thin layer
chromatography-gas chromatography method for the detection of hazelnut oil in
olive oils by determination of esterified sterols
Cercaci L et al., J Chromatogr A 2003, 985, 211
The oil was subjected to SPE, cold saponification and purification on sillica TLC. The sterol band was analyzed by direct GLC. The sterol fraction provides precise information about the origin of olive oil and a possible admixtures with hazel oil.
An important review of published
chromatographic methods for the analysis of plant sterols may be found in the
paper by Abidi SL (J Chromatogr A 2001, 935, 173) and that by Volin P (J
Chromatogr A 2001, 935, 125).
- Capillary electrochromatography of sterols and related steryl esters derived from vegetable oils
Abidi SL, J Chromatography A 2004, 1059, 199
- Quantitative determination of cholesterol, sitosterol, and sitostanol in cultured Caco-2 cells by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.
Palmgren JJ et al., J Chromatogr B 2005, 821, 144-152
- Identification of biologically active triterpenes and sterols present in hexane extracts from Miconia species using high-resolution gas chromatography.
Crevelin EJ et al., Biomed Chromatogr 2006, 20, 827-830
- Rapid and quantitative determination of total sterols of plant and animal origin in liver samples by gas chromatography.
Brufau G et al., Chromatographia 2006, 64, 559-563
- Determination of total sterols in brown algae by Fourier transform infrared spectroscopy.
Bopuzidi N et al., Anal Chim Acta 2008, 616, 185-9
- A simplified method for cholesterol determination in meat and meat products.
Dinh TTN et al., J Food Comp Anal 2008, 21, 306-314
- A simplified method for HPLC-MS analysis of sterols in vegetable oils.
Carretero AS et al., Eur J Lipid Sci Technol 2008, 110, 1142-9
- Application of comprehensive two-dimensional gas chromatography to sterols analysis.
Mitrevski B et al., J Chromatogr A 2008, 1214, 134-142
- Determination of cholesterol in food samples using dispersive liquid-liquid microextraction followed by HPLC-UV.
Daneshfar A et al., J Chromatogr B 2009, 877, 456-460
- A simple and sensitive enzymatic method for cholesterol quantification in macrophages and foam cells.
Robinet P et al., J Lipid Res 2010, 51, 3364-9
- Rapid determination of
cholesterol in emulsified confectioneries by ultra-performance liquid
Ahn JH et al., Eur J Lipid Sci Technol 2012, 114, 1304-11
Structure and nomenclature
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.