Microlipid-induced oxidative stress in human breastmilk: in vitro effects on intestinal epithelial cells.
OBJECTIVE
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 degrees 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.
Diehl-Jones
William L
WL
Faculty of Nursing, University of Manitoba, Winnipeg, Manitoba, Canada. Bill_Diehl-Jones@umanitoba.ca
Askin
Debra Fraser
DF
Friel
James K
JK
D000704:Analysis of Variance; D017209:Apoptosis / Q000187:drug effects; D001681:Biological Assay; D018938:Caco-2 Cells; D020895:Enterocytes / Q000378:metabolism*; D005227:Fatty Acids / Q000494:pharmacology*; D005519:Food Preservation / Q000379:methods; D006801:Humans; D006861:Hydrogen Peroxide / Q000032:analysis; D015850:Interleukin-6 / Q000378:metabolism; D015227:Lipid Peroxidation / Q000187:drug effects; D008895:Milk, Human / Q000276:immunology*; D018384:Oxidative Stress / Q000187:drug effects*; D017382:Reactive Oxygen Species / Q000032:analysis*; D013696:Temperature; D017392:Thiobarbituric Acid Reactive Substances / Q000032:analysis; D013997:Time Factors
D005227:Fatty Acids; D015850:Interleukin-6; D017382:Reactive Oxygen Species; D017392:Thiobarbituric Acid Reactive Substances; D006861:Hydrogen Peroxide