Accurate assessment of n-3 PUFA status and endogenous synthesis is important for determining how these factors relate to health and disease. When measuring fatty acid synthesis it is important to consider the type of tissue and lipid fraction the fatty is being measured in and the measurement technique that is being used. Measurement of fatty acids status and synthesis in humans often requires biological samples that are easy to access and non-invasive, such as blood cells and plasma. The most commonly reported tissues for measuring fatty acids in the literature include subcutaneous adipose tissue from the buttock or abdomen, plasma, platelets and erythrocytes (76). The measurement of a fatty acid in a particular tissue reflects both the intake and the processes
target tissues, metabolism from storage sites or excretion and also endogenous synthesis (77). Exchanges of fatty acids within and between lipid classes in tissues and blood ensures the widespread distribution of fatty acids throughout the body (76). Consequently, measurement of a fatty acid within a particular tissue represents a complex mixture of processes and will include fatty acids that have originated from the diet and from endogenous synthesis.
1.5.3.1 Measurement of n-3 PUFA synthesis using stable isotope tracers
Stable isotope tracer studies provide a powerful approach for measuring n-3 PUFA metabolism in humans, animal models and in vitro systems. The use of 18:3n-3 labelled with stable isotopes such as carbon 13 [13C] or deuterium [2H] has enabled detailed investigations into 18:3n-3 metabolism
including its conversion to longer chain n-3 PUFA (by measuring labelled elongation products) and the extent of β-oxidation (by measuring labelled CO2 on breath or 2H in plasma water). The use of
stable isotopes also overcomes the health hazards associated with using radioisotopes that were traditionally used to measure fatty acid metabolism.
The introduction of gas chromatography (GC) in the 1950s and its subsequent development gave rise to the first convenient means of quantifying stable isotope tracers in complex biological mixtures. This approach relied on the development of sophisticated mass spectrometry (MS) technology techniques. More recently, GC coupled to a combustion furnace followed by isotope ratio mass spectrometry (GC-C-IRMS) has become a popular technique for high precision compound specific isotope analysis of fatty acids. This technique permits measurement of isotope ratios at precisions sufficient to detect differences due to natural processes in all compounds eluted in sufficient quantities from the GC (78). In this process GC separates the analyte from a mixture of compounds, the analyte is then oxidatively combusted and the analyte gases are subsequently ionised and detected allowing the stable isotope ratio to be measured relative to a standard of known isotopic enrichment.
A disadvantage of IRMS is the fatty acid structure cannot be determined as this technique yields no molecular ion data. Furthermore, fatty acids labelled with stable isotopes can be expensive and coupled with the time and effort involved with using stable isotope tracer techniques they become less attractive for use in large-scale studies. Consequently many studies have small numbers of subjects, which raises the question of reliability particularly as individuals often have large natural variability (78). Furthermore, the [13C] content of different foods can vary which could lead to
natural variations between individuals. In order to minimise the impact of this on results, the baseline [13C] enrichment must be subtracted. The average natural abundance of [13C] in food is
1.5.3.2 Measuring n-3 PUFA synthesis using the product to precursor ratio
Another technique that has been used to estimate n-3 fatty acid synthesis and therefore desaturase activity is the ratio of product, for example, 20:5n-3 or 22:6n-3 to precursor (18:3n-3). This ratio is also referred to as the desaturation index. The product to precursor ratio has been used in several studies (79-82) and is well established as a surrogate measure of desaturase activity. It is a particularly popular measurement in studies where it is used as a predictor of metabolic and CVD and mortality (83).
The main advantage of using this technique to measure fatty acid synthesis is that fatty acid profiles and concentrations are relatively simple and cost-effective measurements compared with stable isotope tracer techniques. However, a major issue with this is that the fatty acid composition of the cell membrane is influenced by both dietary intake and metabolic pathways and therefore can only be a crude measure of synthesis. High dietary content of the fatty acid being measured can dilute the pool of endogenously synthesised product therefore causing inaccurate measurement. Furthermore, there is no evidence that this technique has been validated against direct measurements of synthesis using stable isotopes. Su and Brenna (1998), reported a non-tracer method for measuring desaturase activity using GC with flame ionisation detection (GC-FID) (84) . They simultaneously measured delta-6 and delta-9 desaturase activities in microsome preparations using a tracer and non-tracer method and found them to be consistent. However, their measurements were based on the rate of decrease in precursor (or increase in product) over time and were not merely a measure of the product to precursor ratio. A recent study reported a poor relationship between product to precursor ratios and delta-6 desaturase activity and concluded that coupled desaturation-elongation products more accurately reflected activity (22).