Oxidation of marine lipids in liposomes and emulsions mediated by iron and methemoglobin

Abstract

Long chain omega-3 polyunsaturated fatty acids (LC omega-3 PUFA) are vital for physiological functions and have therapeutic and health benefits. The consumption of LC PUFA in the Western world has been below recommended intake levels the past decades, despite promotion of seafood and omega-3 supplements. Incorporation of the LC PUFA into processed food consumed on a daily basis might therefore bridge the gap between the recommended and actual consumption of LC omega-3 PUFA. Unfortunately, the development of omega-3 enriched food is hampered by a very high susceptibility of LC PUFA to oxidative deterioration. Furthermore, oxidised lipids are believed to create health risks. It has also been suggested that gastric juice may deteriorate the healthy LC PUFA after they are ingested. Important lipid oxidation promoters in food are low molecular weight (LMW) iron (Fe) and methemoglobin (metHb). To incorporate the LC omega-3 PUFA safely into food with respect to oxidation, it is necessary to understand both Fe- and metHb-mediated oxidation of PUFA and how the oxidation is influenced by conditions and dietary antioxidants.

The main objective of this thesis is therefore to study Fe- and metHb-mediated lipid oxidation in food-related lipid model systems – emulsions stabilised with phospholipids and liposomes made of phospholipids – containing LC omega-3 PUFA from fish. The focus was on clarifying the reaction mechanisms and the impact of different factors, including dietary antioxidants and gastric juice, on the prooxidant activity of Fe and metHb. Measurement of the consumption rate of the essential substrate for lipid oxidation – oxygen – by the LC PUFA was used for assessment of lipid oxidation.

The continuous measurement of the dissolved oxygen concentration has been shown to be a robust method for direct and instantaneous monitoring of peroxidation in both the liposomes and emulsions. The method was especially useful for measurement of the oxygen consumption kinetics in the lipid systems. The determination of oxygen uptake rates (OUR) enabled screening and evaluation of the impact of the different factors and antioxidants on the prooxidant activity of Fe and metHb.

Pre-formed lipid hydroperoxides (LOOH) were shown to be essential for the prooxidant activity of both Fe and metHb, and the prooxidant activity of metHb was not affected by the lack of light. The oxygen uptake kinetics revealed that iron behaved as a catalyst in lipid oxidation while the prooxidant activity of metHb weakened over time, presumably due to degradation of meHb molecule during lipid oxidation. MetHb was shown to be a stronger prooxidant than Fe, but the strong prooxidative activity was facilitated by a complete structure of the metHb molecule. The prooxidant mechanism of both Fe and metHb was not limited by the level of dissolved oxygen, as long as oxygen was present, or the level of pre-formed LOOH and double bonds in fatty acids, as long as they were present in higher concentrations than the prooxidant.

The extent of the prooxidative activity of Fe was shown to vary in dependence on: The total surface area: Smaller liposomal vesicles with lower lipid content were more prone to oxidation than larger emulsion droplets with a higher lipid content, presumably due to more frequent interactions of Fe with pre-formed LOOH in the interphase.The amount of phospholipid emulsifier: Higher levels of phospholipids resulted in the formation of smaller droplets. The highest OUR were measured for emulsifier concentrations ranging from 5 – 10% (w/w lipid base).pH of the aqueous phase: Fe-mediated oxidation was highest at pH interval 4.5 – 5.5.Dissolved compounds: Sodium chloride (NaCl) and 0.2% of xanthan gum dissolved in the aqueous phase inhibited Fe-mediated oxidation in a concentration dependent manner. Electrostatic retention of Fe by phosphate groups within phospholipid heads has been suggested to facilitate the contact between pre-formed LOOH and Fe, and to create competitive reactions for iron precipitation at pH > 5 and iron complexation by chelating compounds.

The activity of dietary antioxidants has been shown to be affected by the type of prooxidant in the lipid system. Ascorbic acid, caffeic acid, propyl gallate, astaxanthin, ascorbyl palmitate, α-tocopherol, and δ-tocopherol inhibited metHb-mediated oxidation in concentration dependent manners. EDTA had a minor effect on metHb-mediated oxidation.

In Fe-mediated oxidation, caffeic acid, ascorbic acid and α-tocopherol were prooxidants. They directly interacted with Fe, reducing Fe3+ to the more catalytically active Fe2+. The magnitude of the pro-oxidative behaviour was dependent on the Fe-to-antioxidant ratio, antioxidant concentration and pH. Ascorbic acid was depleted by interactions with Fe, and decreased the pro-oxidative activity of α-tocopherol. EDTA and citric acid inhibited Fe-mediated oxidation completely at twice the ratio to Fe and pH > 3.5. Propyl gallate efficiently inhibited Fe-mediated oxidation, while astaxanthin and β-carotene had only minor effects. In addition, chemical structure and physical location of the antioxidants determined their effects.

The work in this thesis shows that for correct interpretation of the effects of antioxidants it is important to assess what types of prooxidants are present in the system.

Both gastric juice and hydrochloric acid solution (HCl) did not prevent oxidation of marine lipids in emulsions and liposomes (pH 4.0). Furthermore, gastric juice did not inhibit metHb-mediated oxidation, but it was capable of reducing the prooxidant activity of dietary LMW iron, compared to HCl solution. Berry juice, green tea, red wine, and caffeic acid reduced the OUR in the acidic environments while coffee, ascorbic acid and orange juice increased the OUR. Therefore, beverages accompanying foods rich in marine lipids will affect the course of post-prandial lipid oxidation.