Microlipid-induced oxidative stress in human breastmilk: In vitro effects on intestinal epithelial cells

Objectives: To (1) determine whether medium chain fatty acids (Microlipid®) added to human breastmilk generates reactive oxygen species (ROS), and (2) measure the physiological effect(s) of Microlipid® (ML)-supplemented human breastmilk in an enterocyte cell culture bioassay. Methods: ML was added to milk according to manufacturer's recommendations and total hydroperoxides measured at intervals with the FOX 2 and TBARS assays. Physiological effects of supplementation were measured using a human enterocyte cell line (Caco-2BBE) and/or a primary human fetal intestinal cell culture (FHS-74 Int). Endpoints included: intracellular oxidative stress, transepithelial electrical resistance (TEER), apoptosis, and interleukin (IL)-6 production. Results: Immediately postsupplementation, ML did not significantly increase ROS, as determined by both the FOX 2 and TBARS assays. Further, storage of milk + ML at 4°C prevented significant increases in total hydroperoxides. However, by 4 hours postsupplementation at room temperature, both assays revealed significantly higher hydroperoxide and lipid peroxide levels. ML-supplemented milk stored at room temperature for 4 hours had the following effects in cell culture bioassays: elevated oxidative stress, increased rates of apoptosis, decreased transmembrane electrical resistance (TEER) values and, in both cell culture assays, significantly increased secretion of IL-6. Conclusions: Based on our measurements of extracellular and intracellular ROS, milk supplemented with fresh ML does not induce significant oxidative stress. However, when stored for 4 hours at room temperature, ML induces significant levels of oxidative stress. Decreases in TEER and increases in apoptosis and IL-6 secretion are consistent with ML-induced oxidative stress. It therefore is likely that in clinical situations, if ML-supplemented milk is not administered quickly, the newborn may be placed at greater risk of oxidative stress.