Detecting Lipid-Derived Electrophiles with a Turn-On Fluorescent Probe. Alkylation of 7-Mercapto-4-Methylcoumarin by α,β-Unsaturated Carbonyls Yields Fluorescence Increase used to Quantify ‘Electrophilic Content’ in Edible Oil Extractions.

Abstract

In the presence of molecular oxygen, edible oils can oxidize to form a multitude of α,β-unsaturated carbonyl products collectively called ‘lipid-derived electrophiles’. These molecules affect the taste of fat-containing foods but also act as electrophiles by covalently binding to protein amines/thiols and DNA nucleotides. The chemical modification of proteins by lipid-derived electrophiles appears to play an important role in human health, but the quantification of this diverse class of compounds remains a challenge. In this study, we describe a method capable of measuring the relative content of α,β-unsaturated carbonyls in food containing edible oils by using a “turn-on” fluorescent probe. The detection of electrophiles is based on a pre-fluorescent probe, 7-mercapto-4-methyl-coumarin (C-SH). The fluorescence of C-SH increases after nucleophilic addition to electrophilic lipid oxidation products. Since different lipid derived electrophiles will react at a different rate with our fluorescent probe, we expressed the probe’s response against a standard electrophile: trans-2-nonenal. In this assay, electrophiles which react more quickly will have a more dominant weight in the measurements carried out. Using this analytical technique, we can compare electrophilic content in French fries from several restaurants, and find they have lower amounts of lipid derived electrophiles versus frozen fries baked at home. We also demonstrate that potato chips sealed in a reduced oxygen atmosphere will have a low ‘electrophilic content’ that increases over time, whereas chips in oxygen-permeable packaging initially have a higher ‘electrophilic content’ that does not increase as much over time. The relative ease of fluorescence measurements using microplate readers coupled with a simple oil extraction protocol should allow this method to quantify ‘electrophilic content’ in several food sources.