Analytical Techniques for Determining Free Fatty Acids in Plasma
Posted by Dr. Theo Kapanadze, D.Sc., Ph.D. on Fri, Mar 30, 2012
The omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to have important physiological effects that may reduce the risk of cardiovascular disease.
It has been proved that the interrelationship between an increased EPA and DHA concentration in blood or red blood cell membrane and the reduced risk of sudden cardiac death. Another risk indicator that has been suggested is the ratio between the omega-6 fatty acid, arachidonic acid (AA) and EPA.
The omega-3 fatty acids found in plasma phospholipids and lipoprotein complexes are a more sensitive indicator of short term changes in the intake of omega-3 over several weeks, whereas the omega-3 fatty acids incorporated in the erythrocyte membrane of red blood cells reflect long-term exposure over several months.
It is recommend an increased intake of EPA and DHA for cardiovascular prevention or during pregnancy. American Heart Association recommends1 g EPA+DHA/d in fish oil or as capsules
However, in natural fish oil, EPA and DHA are bound in triacylglycerides (TAG) whereas many fish oil capsules contain the fatty acids (FA) bound in EE (ethylester) or re-esterified TAG (rTAG). Implicitly, the recommendations assume that different chemical forms of EPA and DHA have comparable bioavailability.
Normally, the recommendations assume that different chemical forms of EPA and DHA have comparable bioavailability.
Recently, has been demonstrated that a six-month supplementation of identical doses of EPA+DHA (1.67 g/d) leads to a faster and higher increase of the omega-3 index - the percentage of EPA and DHA in red cell membranes reflecting the long-term intake and n-3 FA status of an individual when consumed as rTAG than when consumed as EE.
A direct comparison of krill oil to fish oil TAG noted similar changes in plasma FA, while fish oil reduced the blood pressure and krill oil did not.
Traditional analysis of long-chain polyunsaturated fatty acid composition in human blood and plasma requires long and labor-intensive extraction and transesterification methodologies.
Gas chromatography GC-FID or GC–Mass Spectrometry after hydrolysis, extraction and derivatization are the methods of choice, but these methods typically require long run times (>40 minutes) in order to elute the full profile of commonly measured fatty acids. However, methods have been developed by GC analysis where a run time of 11 minutes has been achieved in Krill Oil samples.
For clinical or other biological samples, it is recommended that UPLC-MS/MS methodologies be investigated. Simultaneously quantification of clinically relevant concentrations of EPA, DHA and AA (arachidonic acid) in plasma with a short analysis time (2.5 minutes) has been achieved. For this method, the sample preparation is based on liquid-liquid extraction and the calibration curve is linear with 0.5-300μg/mL using weighted 1/x2 linear regression.
A UPLC-MS/MS method can demonstrate suitable analytical performance characteristics over the currently required concentration range. Measuring EPA, DHA and AA concentrations or calculating the EPA/DHA/AA ratios using this method may facilitate the identification of subjects at potential risk of arrhythmogenic and inflammatory events. Furthermore, intervention to achieve protective concentrations of EPA and DHA by individualized dosing and therapeutic monitoring could be beneficial in reducing the incidence of sudden cardiac death.
