Measurement of conjugated diene lipids by derivative spectroscopy in heptane extracts of plasma

A series of experiments are described which show that second derivative spectroscopy can be used to quantify conjugated lipid dienes as markers of lipid peroxidation in heptane extracts of plasma from patients with rheumatoid arthritis, osteoarthritis, and healthy controls. Results obtained by this method gave reasonable agreement with those derived from the measurement of simple absorbance in chloroform/methanol extracts. Two minima were observed in the derivative spectrum of plasma lipid extracts. These minima occurred at 233 and 241 nm and corresponded to absorbance maxima in the conventional UV spectrum. Using a combination of phospholipase hydrolysis, reverse phase high performance liquid chromatography (HPLC) and second derivative spectroscopy we confirmed that these two minima can be attributed to a single fatty acid (9 cis-, 11 trans-linoleic acid) shown previously to account for greater than 90% of diene conjugation in human plasma samples. When the biological isomer 9 cis-, 11 trans-linoleic acid was separated by reverse phase HPLC from the mixture of other plasma phospholipid-2-esterified fatty acids we observed a change in derivative spectroscopy minima from 233 and 241 nm to 228 and 237 nm. Minima at the latter two wavelengths were also seen with pure preparations of the Paint Research Isomer (9 trans-, 11 trans-linoleic acid) which eluted later than biological 9 cis-, 11 trans-linoleic acid using reverse phase HPLC, suggesting that the absorption spectra of these pure cis-, trans and trans, trans dienes are similar but can be altered by the presence of other fatty acids in the extract.