Relevance of a new HPLC assay for 4-hydroxy-2-nonenal in human plasma

Objective: 4-hydroxy-2-nonenal (4-HNE) is one of the major end-products of arachidonic acid peroxidation since proposed as sensitive oxidative stress marker. Despite the relevance of its physiological and pathogenetic roles, there are currently no available easy-to-use analytical methods for the accurate assay of 4-HNE in human plasma. For this reason, the typical values previously reported were few, contradictory, and often related to small groups of subjects. Methods: We present here a simple and reliable chromatographic method for 4-HNE assay in human plasma. After a deproteinization step, fluorescence derivatization and solid phase extraction (SPE), 4-HNE is finally separated and quantified by a high-performance reversed-phase liquid chromatography (HPLC). Results: An almost complete recovery (> 98%), high sensitivity (limit of detection of 100 pmol/l) and a wide dynamic range (from 1 to 2000 nmol/l) propose this method as particularly suitable for high throughput measurements of 4-HNE in human plasma samples. The typical values of 4-HNE (37 ± 15 nmol/l, mean ± SD) were measured in a group of 96 elderly volunteers. Conclusions: This simple HPLC method, with its good SPE cleaning and high sensitivity of detection, is proposed as a very reliable and advantageous tool to characterize lipid oxidative damages in several chronic pathological conditions. The typical plasmatic levels measured were very little scattered but much lower than those previously reported by other authors. This discrepancy suggests that, in some cases, it has been analyzed not only the free, but even the bonded fraction of 4-HNE.