Fatty acid analysis

2.6.1. Extraction. Total fatty acids were extracted using Chloroform:Methanol (C:M; 2:1 v/v) following Folch (1957). Animals were homogenised using a micro-mortar and pestle to ensure complete extraction. The mortar and pestle was rinsed with 1 ml solvent (C:M; 2:1 v/v), which was then added to the sample. All samples were topped up with solvent (C:M; 2:1 v/v) until they were exactly 2 ml and transferred into clean 4 ml vials. An additional ‘blank’ vial (4 ml) was filled with 2 ml of solvent (C:M; 2:1 v/v) and treated exactly as the other samples. To accurately quantify the amount of fatty acid present, a known amount of

21:0 fatty acid internal standard was added to each sample (2.5 µg to eggs and

particulates and 5.0 µg to animals).

The non-fatty acid fraction (sugars, urea, amino acids and salts) was removed

using phase separation by adding 500 µl of 0.88% KCl. Following whirlimixing

(vigorous shaking) and centrifugation (2 mins at 1500 rpm), the top aqueous layer (containing the non-fatty acid fraction) was removed and discarded. The organic layer (containing sample) was evaporated under a constant flow of oxygen free N gas. Any water remaining was subsequently removed by drying the samples under vacuum in a desiccator containing pre-combusted silica-gel.

2.6.2. Alkaline hydrolysis (saponification). This process is primarily to produce free fatty acids, although it also serves to remove some of the unwanted long

chain alcohols and sterols. The free fatty acids are produced by adding 500µl of 1M

KOH in 95 % ethanol to the dry vials (containing sample) and maintaining them at

acidified with a few drops of 0.6M HCl. The free fatty acids were then extracted by

two sequential washes with diethylether; after adding 1000 µl of diethylether (500 µl

on the second wash), the sample is whilimixed and centrifuged. The upper layer (containing sample) is transferred to a clean 2 ml via. The sample was then evaporated under N gas and dried by vacuum desiccation.

2.6.3. Preparation of PFB esters (fatty acid derivatization). The samples for fatty acid analysis e.g. seston and eggs, contained only small quantities of fatty acids. Therefore, rather than using a Flame Ionisation Detector (FID), the gas chromatograph (GC) was fitted with a highly sensitive electron capture detector (ECD). By halogenating the free fatty acids with pentafluorogenzyl (PFB) estetrs, nanogram quantities of fatty acids could be detected.

After saponification, the free fatty acids were dissolved in 30 µl of

acetonitrile and agitated. Following this, 100 µl of 2,3,4,5,6-pentafluorobenzyl

bromide (PFP-Br) solution was added and similarly agitated. Finally, after adding

100 µl triethylamine and mixing, the samples were agitated and left to derivitize for

15 minutes at room temperature. The fatty acids were extracted by two sequential

washes with isooctane (500 µl). The upper layers, containing the PFP esters, were

transferred to a clean 2 ml vial. The sample was then evaporated and dried before being redissolved in isooctane.

2.6.4. Purification. The fatty acids were separated from any remaining contaminants using high performance thin layer chromatography (HPTLC). The silica HPTLC plates (10 x 10 cm) were pre-run, using an 18 ml hexane, 2 ml

diethylether and 200 µl acetic acid solvent system. The PFB esters were dissolved in

100µl isooctane and applied to the HPTLC plates via a syringe, alongside a PFB

ester standard. Upon completion of the chromatographic separation, the PFB ester standard was labelled with 2,7-dichlorofluorescein (DCF) dissolved in methanol (Christie 1973) and the band visualised under ultra-violet light. The area of silica corresponding to each sample of purified PFB esters was scraped off the plate using a scalpel blade, and transferred to clean 8ml vials containing 2 ml isooctane. After

the addition of 1 ml NaHCO3 (2 % W/V) the sample is whirlimixed, centrifuged and

frozen at –20 ºC. The upper, non-frozen, isooctane layer was transferred to a clean 2 ml vial and evaporated under N gas. The samples were dissolved in isooctane and stored in 1.1 ml pear shaped vials at –20 ºC until required for injection. The volume of isooctane used depended on the quantity of fatty acid within the sample. The ideal

loading value for gas chromatography was 0.2 µg of fatty acid per µl injected, but without knowing the absolute values of fatty acid within the sample, a degree of trial and error was required to find the correct volume of isooctane.

2.6.5. Injection and identification of fatty acids. Fatty acid PFB esters were analysed by gas chromatography coupled with an electron capture detector (GC- ECD), using a ZB-Wax 30 m x 0.32 mm internal diameter column and hydrogen as the carrier gas. The oven temperature program was as follows; 80 ºC to 190 ºC at 40

ºC min-1, 190 ºC to 230 at 4 ºC min-1, remaining at 230 ºC for 47 minutes. Because

the retention times of the column vary slightly over time, a marine fish oil standard (Marinol) containing a full suite of fatty acids, was injected at the start of every day. Individual fatty acids were identified by comparing retention times of those identified on the Marinol trace using ThermoFinnigan Chrom-Card software to those in the sample.

2.6.6. Quantification of fatty acids. The electron capture detector (ECD) quantifies each individual molecule of a particular fatty acid. The corresponding fatty acid peak on the trace is thus directly related to its quantity (moles). Because a known quantity of standard (21:0) was added, the quantity (moles) of any identified fatty acid can be determined using the following relationship:

M A A M S S 21:0 0 : 21 = (5)

where S is the identified fatty acid, M is the quantity in moles, and A is the area of the

respective peaks. The absolute quantity (µg of lipid) of a particular fatty acid, SQ, can

then be determined:

SQSAM (6)

where S is the particular fatty acid and AM is the atomic mass (in µg).

2.7. Statistical analysis. Parametric statistics can only be used to compare samples