The aim was to extract fatty acids that had not been incorporated into Ca-fatty acid soaps,
including free fatty acids, Na-fatty acid soaps, neutral fats (eg. mono, di, triglycerides) and
phospholipids from faeces, leaving the Ca-fatty acid soaps remaining in the faeces which
could then be determined using fatty acid analysis. Solvents with the lowest Ca-fatty acid
soap solubility, as determined in section 3.3.3, were chosen for the extraction of non-Ca
bound fatty acids. The sequential three-step extraction method was performed as follows
(Figure 3–1): Step 1) extraction with hexane (a non-polar solvent) to remove free fatty acids
and triglycerides; step 2) extraction with hexane-isopropanol (a more polar solvent mixture)
to remove neutral fats (eg. diglycerides, monoglycerides, phospholipids) (the latter solvent
mixture was proposed by Hara and Radin (1978) as being suitable for the extraction of polar
Chapter Three: Assay Development for Ca-Fatty Acid Soap Determination
68
Figure 3-1: Sequential three-step solvent extraction of non-Ca bound fatty acids from faeces containing free fatty acids, mono-, di-, and triglycerides, phospholipids, Na-soaps and Ca-fatty acid soaps.
Step 1: Extraction with hexane to remove free fatty acids from the faecal matrix
Hexane (3 mL) was added to all of the faecal samples and mixed using a vortex mixer to
allow extraction of the free fatty acids. Of the four replicate samples, one duplicate set(set A)
was kept at room temperature for 10 min while the second duplicate set (set B) was
incubated at 60qC for 10 min. After incubation, the tubes were again mixed on a vortex
mixer. Each tube was then centrifuged at 1500 g for 5 min and each supernatant transferred
into a separate screw cap glass tube. Each of the precipitates was washed twice with 2 mL
hexane, with the washings being pooled with the supernatant for each sample separately.
The solvent was then removed from each tube by evaporation under a stream of nitrogen.
The extracted free fatty acids were then determined as described in section 3.3.5. The
Chapter Three: Assay Development for Ca-Fatty Acid Soap Determination 69
Step 2: Extraction with hexane-isopropanol to remove polar lipids from the faecal matrix
A mixture of hexane-isopropanol (3:2) (3 mL) was added to the faecal precipitate from the
previous hexane extraction step. Samples were extracted with hexane-isopropanol for 15 min
where again one duplicate set (set A) was kept at room temperature and the second
duplicate set (set B) was incubated at 37qC. The samples were centrifuged at 1500 g for 5 min
and each supernatant transferred into a separate glass tube. The extraction step was repeated
and the supernatant pooled with the previous supernatant for each sample separately. The
extracted free fatty acids were then determined as described in section 3.3.5. The faecal
precipitates were dried under a stream of nitrogen to remove any residual hexane-
isopropanol prior to commencing step 3.
Step 3: Aqueous extraction of Na-soaps from the faecal matrix
Distilled water (6 mL) was added to the tubes containing the faecal precipitates that had
been previously extracted with hexane and hexane-isopropanol. The tubes were mixed on a
vortex mixer and incubated for 30 min. Duplicate set A was kept at room temperature,
whereas duplicate set B was incubated at 80qC. After incubation, all the samples were
filtered through a Buchner funnel (previously heated to 95qC for the duplicate set B) attached
to a vacuum pump. All the tubes were quantitatively washed with distilled water either at
room temperature for duplicate set A or water heated to 80qC for duplicate set B. The
amounts of Na-fatty acid soaps in the filtrate were then analysed by firstly acidifying the
filtrate, extracting with hexane and then determining the extracted fatty acids as described in
section 3.3.5. The residue present on each filter disc containing the insoluble Ca-fatty acid
soaps, was dried overnight, inserted into screw top glass tubes and prepared for fatty acid
Chapter Three: Assay Development for Ca-Fatty Acid Soap Determination
70
Preparation of model faecal samples to test the efficacy of the three-step extraction method for removing non-Ca bound fatty acids
Model faecal samples containing known amounts of free fatty acids, Na-fatty acid salts,
neutral fat and Ca-fatty acid soaps comprising medium chain saturated, long chain saturated
and long chain unsaturated fatty acids were prepared. Defatted faecal samples (treated as
described in section 3.3.1) were spiked with either: 1) Ca-laureate, Na-stearate and free oleic
acid; 2) Ca-stearate, Na-oleate, and free lauric acid; 3) Ca-oleate, Na-laureate and free stearic
acid. An additional faecal sample was spiked with phospholipids (Sigma-Aldrich) to
evaluate the efficacy of the three-step extraction method for removing phospholipids. The
defatted freeze dried faeces (50 mg) were weighed in quadruplicate into screw cap glass
tubes. Ca-fatty acid soaps, Na-soaps and free fatty acids (approximately 3 mg of each) were
added and mixed into the faecal samples. To spike the faeces with phospholipids, 3 mg of
the phospholipids were dissolved in distilled water (1 mL) and 50 mg of defatted freeze
dried faeces were added, mixed and dried overnight under vacuum at 37qC. In addition,
faeces containing the added phospholipids were also spiked with 3 mg of Ca-fatty acid
soaps, Na-soaps and free fatty acids as described above to investigate the behaviour of
Ca-fatty acid soaps, Na-soaps and free fatty acids in the presence of phospholipids during
Chapter Three: Assay Development for Ca-Fatty Acid Soap Determination 71
3.4.
Results & Discussion
3.4.1.
Purity of synthetic Ca-fatty acid soaps
The purity of the synthetic Ca-fatty acid soaps was assessed based on the determined molar
ratio of Ca to fatty acids. The molar ratio of Ca to fatty acids in the Ca-fatty acid soaps
synthesised with lauric, myristic, palmitic, stearic, oleic or linoleic acid after purification is
shown in Table 3-1. The determined molar ratios were generally close to
the expected ratio of 1:2, indicating that
soaps had been formed. The poorest ratio
was observed for the Ca-linoleic acid soaps,
which had a molar ratio of Ca to fatty acids
of 1:1.6. The lower ratio may indicate
incomplete soap formation or may be due
to the oxidation of linoleic acid as it can
oxidise quickly when exposed to air.
Table 3-1: Determined molar ratio of Ca to fatty acids in the synthetic Ca-fatty acid soaps1.
Fatty acid present in the soap
MW2 Ca:fatty acid molar
ratio Lauric acid 200.32 1:1.8 Myristic acid 228.38 1:2.1 Palmitic acid 256.42 1:2.0 Stearic acid 284.48 1:1.9 Oleic acid 282.46 1:1.8 Linoleic acid 280.45 1:1.6 1The molar ratio of Ca to fatty acids in the synthetic soaps was analysed in duplicates at three different times; mean values (n=3) from fatty acid analysis and Ca-analysis were used to calculate the molar ratio of the synthetic soaps.
2molecular weight (MW) of the individual fatty acids