Polyunsaturated Fatty Acids and Their Therapeutic Functions

MTB Lecture # 9

PUFA Many of the fatty acids can be synthesized by humans, but there is a

rgoup of PUFA, the essential fatty acids, that the human body cannot produce: omega-3 (n−3) and omega-6 (n−6) fatty acids.

The parent omega-6 fatty acid is linoleic acid (C18:2n−6, LA) and the parent omega-3 fatty acid is α-linolenic acid (C18:3n−3, ALA).

Omega-6 fatty acids as arachidonic acid (C20:4n−6; AA) can be synthesized by humans from LA, and omega-3 fatty acids, as eicosapentaenoic acid (C20:5n−3; EPA), docosapentaenoic acid (C22:5n−3, DPA) and docosahexaenoic acid (C22:6n−3, DHA), from ALA.

The conversion of ALA in EPA, DPA and DHA is low and these omega-3 fatty acids are considered essential fatty acids too. Therefore, both n−3 and n−6 PUFA are entirely derived from the diet and necessary for human health.

Omega-3 fatty acids are a class of polyunsaturated fatty acids. They all have a double carbon-to-carbon bond in the third position from the omega (or methyl, or n) end of the fatty acid chain.Omega-3 family include

Eicosapentaenoic acid (EPA) Docosahexaenoic acid (DHA) Alpha-linolenic acid (ALA).

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PUFA Both n−3 and n−6 PUFA are entirely derived from the diet and necessary

for human health.

An n−6:n−3 fatty acid ratio of 5:1 or less is desired, as suggested by nutrition experts (WHO/FAO, 1994).

Modren trends in food a high consumption of meat, seed oils, fast food (pizzas, hamburgers…) and snack food (cakes, biscuits…), that contain a large amount of saturated fatty acids and a low proportion of PUFA (Fernández-SanJuan, 2000).

The researches concluded that the n−6:n−3 ratio in blood, in people who consume Japanese or Mediterranean food, is close to 2:1, while in people who consume fast food, it can reach values up to 25:1, much higher thandesired

EPA and DHA EPA and DHA are found primarily in fatty fish; ALA is abundant in

flaxseed, walnuts, and soybeans.

The human body can convert small amounts of ALA into EPA and DHA: only about 5% of ALA is converted to EPA and less than 0.5% isconverted to DHA.

It is not knownwhether ALA is active itself or only via these metabolites

Healthy people should consume fish (preferably oily fish) at least twice a week, according to the American Heart Association

cod and catfish contain 0.2 g of EPA/DHA per 100-g serving; others, such as Atlantic salmon, contain about 10 times as much

Omega-3 fatty acids and human healthThe omega- 3 index (percentage of EPA+DHA of total fatty

acids in red blood cells) as a risk factor for sudden cardiac death should be higher than 8%. There are also several studies that propose the mechanisms by which the omega-3 PUFA act in humans

Fish oil supplements lower triglyceride levels and may have other benefits such as preventing arrhythmias, reducing inflammation (although they have minimal impact on C-reactive protein), inhibiting platelet aggregation, and lowering blood pressure, all of whichshould reduce cardiovascular risk.

Hypertriglyceridemia is thought to increase the risk of coronary heart disease by two mechanisms.

Firstly triglyceride-rich lipoproteins such as verylow-density lipoprotein (VLDL) and intermediate-density lipoprotein (IDL) are thought to be atherogenic.

Secondly, triglyceride-lipoprotein metabolism involves

competition with high-density lipoprotein (HDL), leadingto a decrease in HDL production and to denser LDL particles.

Omega-3 fatty acids and human health

How omega-3 fatty acids lower triglyceridelevels One mechanism, seen in animal studies, is by decreasing

hepatic synthesis and secretion of VLDL particles by inhibiting various enzyme transcription factors.

Another proposed mechanism is that EPA and DHA increase the activityof lipoprotein lipase, leading to an increase in chylomicron clearance.

This was validated by Khan et al (2011)who showed that lipoprotein lipase activity increased in patients who received omega-3 fatty acids 3 g/day for 6 weeks.

How much do they lower triglyceridesData from the makers of Lovaza3 indicate that in a patient

population with a mean baseline triglyceride level of 816 mg/dL, 4 g/day of omega-3 fatty acids lowered triglyceride levels to 488 mg/dL, a 45% reduction (P < .0001).

In addition, HDL cholesterol (HDL-C) levels increased by 9%.

Higher the base line more efective is this conversion.

People with known coronary artery disease should take in 1 g of EPA/DHA per day according to the American Heart Association

Procedures to obtain long chain omega-3 PUFA

The procedures to obtain long chain omega-3 PUFA concentrates, usually from the natural sources that contain them, have been investigated and some new procedures are being proposed.

Production of omega-3 fatty acids from fish:

The most important natural sources of omega-3 PUFA are marine organisms (fish, seafood, algae), that are fed, directly or indirectly, from marine phytoplankton, the primary producer of omega-3 in the trophic chain

In general, traditional processes to obtain fish oil involve two stages:

oil extraction from raw material and refining.

Some other new processes to obtain omega-3 concentrates for pharmaceutical or nutritional purposes include enzymatic methods and methods that use supercritical fluids.

Traditional Fish oil extraction processes Fish oil is produced from the antiquity by Nordic towns that used it

as fuel in lamps. At the beginning of the 19th century, USA began to produce fish oil from menhaden, using a process with two steps:

Fish cooking and rock-weighted pressing later this press was replaced by mechanical screw presses and later by hydraulic presses.

To improve the yield of the extraction, the quality of the fish oil extracted and the profitability of the process for industrial purposes, especially using fish by-products as raw material

The traditional methods that use organic solvents for oil extraction are widely applied for analytical purposes but not for production due to the several drawbacks of using solvents with restrictions in the food

wet pressing method Nowadays, the most common process to obtain crude fish

oil from fresh fish at industrial scale is the wet pressing method, as describedby the Food and Agriculture Organization of the United Nations (FAO,1986).

This process involves the production of fish oil and fish meal through several steps, i.e. cooking of the raw material, pressing of the cooked material and final filtration or centrifugation to recover the oil from the miscella. The use of a 3-phase centrifuge can greatly simplify the separation stages after cooking.

Different fish by-products have also been proposed as sources of fish oil such as separation of oil from tuna heads.

Flow diagram of the conventional method to obtain crude fish oil

Supercritical fluid extraction (SFE) The fluid possesses intermediate properties between a gas and a liquid, i.e.:

liquid-like density and gas-like viscosity and diffusivity. That is, supercritical fluids (SCF) have at the same time a good solvent power and good transport properties

The mostwidely used SCF is carbon dioxide that is considered a green solvent; CO2 is non toxic, cheap and non flammable. As is gaseous under ambient conditions and therefore easy to separate fromthe processed products after processing.

Fish oil solubility increased significantly with pressure, whereas, at constant pressure, oil solubility decreased with temperature up to a pressure value from which oil solubility began to increase as temperature increased.

Solubility of fish oil from sardine in SC-CO2 in a pressure range of 20 to 35 MPa at four different temperatures (313, 333, 343 and 353 K)

The extraction of sardine oil with SCCO2, concluded that it is possible to find the conditions to recovermost of the oil (95%) without degradation of the omega-3 PUFA

Oil extraction by enzymatic methods

This technology requires neither organic solvents nor high temperatures

The use of enzymatic technology to release lipids from fish avoiding the use of solvents and high temperatures: Liaset, Julshamn,&Espe (2003) studied the enzymatic hydrolysis of salmon frames with proteases and the composition of the different fractions obtained after separation by centrifugation.

They reported that this process enables to obtain omega- 3 enriched oil with a good recovery (about 77%) as well as several interesting products such as peptides or essential amino acids.

Linder, Fanni, and Parmentier (2005) developed another enzymatic method to extract oil from ground salmon heads at middle temperature (55 °C) different commercial enzymes: a protease (Alcalase), an exopeptidasen(Neutrase) and an endo-peptidase (Flavourzyme).

Fish oil refining The oil extracted from a natural material is a mixture of several compounds as

free fatty acids, glycerides, phospholipids, sterols, pigments or tocopherols, and, sometimes, toxics such as heavy metals, dioxins or PCBs (Cheryan, 1998).

Oil refining needs to be performed to remove non-triglyceride, colorants, smelly and toxic compounds in the production of edible oils.

The conventional oil refining in industry is usually made by chemical methods, which include several steps

degumming, to separate phospholipids neutralization or deacidification, to clear free fatty acids and

decrease oil acidity bleaching to absorb pigments or contaminants and d eodorization to remove smelly compounds.

This process presents several drawbacks since it involves the use of chemical products (alkalis) that contaminate the environment and some neutral oil is lost, mainly in oils with high free fatty acid content

Physical refining processes, based on the application of superheated steam under low pressure have been

proposed as alternative to remove free fatty acids and volatile compounds.

However, these methods require a preliminary step of chemical refining and, due to the use of high temperatures, they are not suitable for thermolabile oils such fish oil.

Physical adsorption on activated carbon has been proposed recently to remove contaminants, such as dioxins and PCBs, from fish.

Traditional oil deodorization is based on the application of high temperatures is not good for fish oil above 180 C the an important PUFA degradation, involving the formation of many undesirable compounds such as polymers, PUFA isomers, mono and di-trans and cyclic fatty acid monomers.

vacuum steam distillation at low temperatures followed by a treatment in a silica gel column , adsorption with a resin or treatment with diatomaceous earth have been proposed for removing smelly compounds from fish oil.

Supercritical fluid technology, together with membrane and enzymatic processes, is one of the most recent technologies proposedas alternative to oil refining with chemical products or high temperatures.

Omega-3 concentration from fish oil Enzymatic methods in omega-3 concentration Stabilization of omega-3 PUFA Alternative sources of omega-3 fatty acids Medical applications

Omega-3 concentration Omega-3 fatty acids are better absorbed by human organism when they

are as acylglycerides than as esters and their stability against oxidation is also higher when omega-3 are as acylglycerides than as estersEnzymatic methods in omega-3 concentration

Normally lipases, which are able to catalyse reactions such hydrolysis, ethanolysis or transesterification of triglycerides.

Due to the fatty acid distribution in the glycerol backbone found in severalmarine triglycerides (Ando et al., 1992) and the stereospecifical activity of certain lipases (Wong, 2003), these methods are very useful both in omega-3 concentration from fish oil and in the production of structured lipids.

In the last years, a great amount of enzymatic methods followed by a separation process such membrane filtration, urea complexation or molecular distillation have been proposed in the literature to obtain omega-3 concentrates, especially EPA and DHA, as different forms such free fatty acids, ethyl esters or 2-acylglycerides

Alternative sources of omega-3 fatty acids The benefits of fish as source of omega-3 are sometimes

questioned (Domingo, 2007) and several alternative sources for omega-

3 PUFA have been explored.

Marinemicroalgaes, algaes or transgenic plants. A study comparing microalgae oil and fish oil concluded that both oils present a similar amount of omega-3, although microalgae oil has the advantage of presenting neither an unpleasant odour nor a high amount of cholesterol, and contains squalene and phytosterols, which offer additional benefits to human health.

In addition, microalgaes are easily cultivated, which avoid differences in seasonal production and enables increasing the productivity of PUFA from an industrial point of view.

two main techniques used at the time for obtaining highly pure PUFA: urea fractionation and HPLC, and detailed what were “potentially useful techniques” such as supercritical fluid extraction and lipase-catalyzed processing.

From then to now, several authors have proposed new PUFA extraction methods from different microorganisms, many of them using supercritical fluid technology, similar to the fish oil extraction processes.

Transgenic plants have been also proposed as an alternative source of omega-3 fatty acids. Napier (2006) has recently published a review with the last advances in the production of enriched vegetables species through genetic modifications. Robert (2006) has also compiled the most recent studies about production of transgenic seed oils and their use in human and aquaculture nutrition.