A miniaturized iodine value assay for quantifying the unsaturated fatty acid content of lipids, lipid mixtures, and biological membranes

Various methods exist for identifying and quantifying lipid unsaturation, including mass spectrometry and Raman spectroscopy. A disadvantage of these existing approaches is the need for sophisticated equipment and software, placing them beyond the means of many laboratories. The iodine value (IV) is a colorimetric unsaturation index; however, it uses iodine monochloride, a hazardous chemical, and considerable amounts of sample. Here, we demonstrate the first use of a miniaturized IV method that requires only milliliter quantities of hazardous chemicals and sample sizes such that it is feasible to assay biological membranes. Briefly, lipids are exposed to iodine monochloride, resulting in the replacement of unsaturated bonds with di-halogenated single bonds. Potassium iodide then reacts with unreacted iodine monochloride forming I₂, which is quantified through titration with sodium thiosulfate. To demonstrate the biological relevance of our assay, membrane lipids of Escherichia coli grown at 30, 37, and 42°C were analyzed, with IV increasing as temperature decreased, as would be expected. Importantly, multiple samples could be rapidly and simultaneously analyzed in a reproducible assay that did not require sophisticated equipment or data analysis methods. Our miniaturized IV assay will benefit laboratories with limited access to sophisticated equipment and enable the rapid determination of lipid unsaturation in milligram-scale samples.