desmethylsterols.9 be hydrolysed under basic conditions. Fractionation

desmethylsterols.9 Steryl glycosides will be
discarded with the aqueous phase as they won’t be hydrolysed under basic
conditions.

 

Fractionation of
total lipid and non-polar lipid is mainly done by column chromatography (CC) on
silica gel or alumina but thin-layer chromatography (TLC) is preferred for
small amounts. Steryl conjugates such as steryl esters28, steryl glycosides29 and acylated steryl glycosides30 can all be separated using these
techniques. Before the fractionation of individual sterol subclasses, CC is
used to separate the polar lipids from non-polar lipids e.g. non-polar lipids
are eluted from a silica-gel column with chloroform and polar lipids are eluted
with methanol.31

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Purification

 

Over a century ago, Windaus32 made a discovery that
some sterols, such as sitosterol and ß-cholestanol, can form a complex with the
plant saponin, digitonin, which forms a precipitate whereas ?-cholestanol
and cholesterol esters do not. From the precipitate the free sterols can be
recovered and redissolved for TLC.11 The formation of this
sterol-digitonin complex (also known as digitonide) has been used as a
purification step to separate steroids of the beta configuration from those
with the alpha configuration.33 However, digitonin is
rarely used now as it is poisonous and expensive. Also, it has been outdated by
many new and improved chromatographic procedures for sterol purification. The
ease of the precipitation depends on factors such as length of side chain, function
of sterol configuration at C-5 and C-17 and presence or absence of D-ring
subsitutents.13

 

 

 

To quantitate and characterise sterols, a
series of chromatographic separations produce pure compounds that are further
evaluated by spectroscopic techniques. The proof of the sterical purity of the
chiral alkyl group is achieved by nuclear magnetic resonance (NMR) spectroscopic
techniques.29 GC-MS, ?-values and sometimes 1H NMR are
used to identify the sterols.

 

The preliminary
aim is to recover a fraction from the lipid that is rich in a sterol, a mixture
of closely-related sterols or a group of sterol conjugates. This fraction is
then analysed and the data is used to decide whether further purification is
necessary. Compounds such as triacylglycerols, wax esters, phospholids and
other esters can interfere with chromatography and majorly contribute to the
bulk of the fraction so it best to destroy these by saponifcation.9 Saponification of the
total lipid can usually lower the mass of an oily liquid by 70-80% to yield a
solid non-saponifiable fraction which contains sterols. Having a much smaller amount
of material makes chromatographic procedures simpler.

 

Knowing
the distribution of sterols and between the free sterol and various steryl
conjugates is very useful as it provides information for the biochemistry and
functions of sterols in an organism. This can be done by… steps on page 70 of
analysis of sterols)

 

The most common method for isolating sterol
esters is preparative TLC, often with a preliminary purification step. 34 However, a problem with this method is that there can be
a slight contamination of the sterol ester fraction due to triacylglycerols being
greater in weight and having similar polarity.

 

A more efficient method is preparative
high-performance liquid chromatography (HPLC).

 

Use of a non-destructive detector to track the eluting compounds
allows collection of the separated fractions.

 

Evershed et al. (1987) used column chromatography on alumina to obtain
a fraction containing the sterol esters followed by either preparative TLC or
Sep-Pak cartridges to isolate the sterol esters from the mixture.

 

 

A problem with HPLC is the lack of resolution of different steryl
ester. This makes it difficult to assign unambiguous identifications of
components in complex mixtures. This is particularly the case with steryl
esters of plant origin, which may contain several different sterols, in
contrast to the mammalian steryl esters, where cholesterol may be the major or
only sterol component. Another major disadvantage of HPLC for the analysis of
steryl esters lies in the relatively poor detection limits. The lack of a
strong chromophore means that 10-50 fig of each component is generally required
for analysi9. 35

An
alternative to HPLC which overcomes some of these difficulties is the analysis
of steryl esters by GC and GC-MS.

GC
is a considerably more sensitive analytical procedure than HPLC for the
investigation of steryl esters li-18. Moreover, GC has a major advantage that
it is easily interfaced to a mass spectrometer, thus providing the opportunity
for more certain compound identification ‘

Because
of the relative involatility of steryl esters, a short column is required with
good thermal stability for temperature programming up to about 350°C. We have
found that the most suitable columns for our purposes are 8-12 m x 0.22 mm
flexible fused-silica capillaries with

 

 

The
sterols were isolated by preparative TLC (silica gel, chloroform-ethanol, 98:2)
36

 

 

GLC is the
preferred method because of it’s greater specificity and accuracy. It can
distinguish between cholesterol and the closely linked phytosterols for example
ß-sitosterol, campesterol and
stigmasterol etc.37

 

The circulating form of sterols in humans is
primarily as steryl esters, in which a fatty acid is esterified to carbon 3 of
the sterol; however, a small variable percentage of free sterols also
exist. 

This duality poses additional analytical challenges
because free versus esterified sterols must be isolated or measured separately,
or steryl esters must first be converted to free sterols. 27

 

 

 

Dried
material extraction or wet extraction combined with saponification is the
technique used regarding stools38. Börgstrom39 developed a method to isolate sterol esters from human faeces and
Fillerup and Mead40 developed methods to extract from animal tissue. They were able to
separate cholesterol esters from triglycerides using silicic acid.

– Use a
modified technique for the lipids of human faeces and able to isolate cholesterol
ester fractions. Using G-LC, examine the fatty acids present in the esters.

–       Very small amount of sterol esters in human faeces (necessary to extract
large quantities of material) – read more of 11.

 

 

We have applied a modification of this technique to the
lipids of human fæces and have been able to isolate cholesterol ester
fractions; and, using gas-liquid chromatography, have examined the fatty acids
present in the esters.