Oxidation of marine oils during in vitro gastrointestinal digestion and its effects on stress in human intestinal Caco-2 cells

Doctoral thesis, 2017

Marine oils are attracting public interest due to the preventive effects, e.g., on inflammation, which are linked to the long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFAs). However, LC n-3 PUFAs are highly susceptible to oxidation, which could interfere with their positive effects. It has been shown in vitro that marine lipids not only oxidize during storage, but also during gastrointestinal (GI) digestion. Little is so far known about the marine lipid oxidation reaction under human GI conditions. In this work, oxidation of marine oils during in vitro GI digestion was investigated targeting the highly reactive lipid oxidation products malondialdehyde (MDA), 4-hydroxy-trans-2-hexenal (HHE), and 4-hydroxy-trans-2-nonenal (HNE); all three with documented carcinogenic and genotoxic properties. Variables studied during the digestions were; source of the GI-fluids (porcine/human), presence of additional gastric lipase (from rabbit) or addition of a lipase inhibitor, type of in vitro model (static/dynamic),physical status (bulk/emulsified), oxidation status and origin of the marine oil, as well as additions of food-derived pro- and antioxidants. Furthermore, effects from marine oil digests related to intestinal cell stress were studied. Aldehyde levels increased over time in the intestinal phase during digestion of cod liver oil, in a static in vitro digestion model with human digestive fluids (HDF) or simulated digestive fluids (SDF, i.e., electrolyte solution with enzymes and bile of porcine origin). The highest aldehyde levels were reached during the intestinal phase (t=210 min) using HDF (60 µM of MDA, 0.96 µM of HHE, and 1.6 µM of HNE). In the static model with HDF, lipolysis was found to correlate positively to lipid oxidation, as shown when adding rabbit gastric lipase or orlistat, a lipase inhibitor, to cod liver oil. Aldehydes also increased during digestion of cod liver oil in a dynamic digestion model (tiny-TIM) with SDF. Cod liver oil having a higher degree of oxidation at start of the digestion reached higher levels of aldehydes during GI conditions compared to non-oxidized oils. Pre-emulsification of cod liver oil was slightly protective in the gastric phase, but had a pro-oxidative effect during the intestinal phase. Addition of fish hemoglobin (Hb) as a pro-oxidant to emulsified cod liver oil strongly promoted aldehyde formation, while the metal chelator EDTA had a protective effect during gastric digestion. Industrially relevant levels of tocopherols (α-tocopherol, and Covi-ox® T 70 EU; 4.5 mg/g oil) were protective to cod liver oil oxidation in the static in vitro digestion model with HDF. In the same model, detected aldehyde levels in intestinal digests from four different marine oils were ranked as: cod liver oil ~ whole fish oil >> krill oil ~microalgae oil.  To study cellular effects of GI oxidation, a cultured human intestinal epithelium (Caco-2 cell line) was treated with cod liver-, fish-, and algae oil digests, and corresponding levels of pure MDA and HHE (0-90 µM). Cell viability was not affected by the digests, nor their levels of MDA and HHE. Stress-related proteins were not found to increase upon exposure to digests or aldehydes, rather the opposite. To summarize, MDA, HHE, HNE were formed during in vitro GI digestion of marine oils in all the models tested; absolute levels were, however, affected by pre-treatment of the oils, and were higher with HDF than SDF. Although bulk oils digested without added pro- or antioxidants did not induce a stress response in the Caco-2 cells, studies in humans are needed to be able to say if the absence of stress effects from aldehydes or other oxidation products can be translated to in vivo conditions.