Quantification of 4-hydroxy-2-nonenal (HNE) bound to circulating proteins may prove to be useful in evaluating the role of this bioactive lipoperoxidation by-product in the pathogenesis of various diseases. Recently, we developed a quantitative gas chromatography–mass spectrometry (GCMS) assay of total protein-bound HNE (HNE-P) in blood after reduction with NaB2H4 and cleavage with Raney nickel. Whereas it has been assumed that Raney nickel cleaves only Michael adducts of HNE to cysteine via a thioether bond (HNE-SP), results from this study demonstrate that our GCMS method also detects with precision picomoles of HNE adducts via nitrogen residues (HNE-NP). Specifically, evidence was obtained using variou... More
Quantification of 4-hydroxy-2-nonenal (HNE) bound to circulating proteins may prove to be useful in evaluating the role of this bioactive lipoperoxidation by-product in the pathogenesis of various diseases. Recently, we developed a quantitative gas chromatography–mass spectrometry (GCMS) assay of total protein-bound HNE (HNE-P) in blood after reduction with NaB2H4 and cleavage with Raney nickel. Whereas it has been assumed that Raney nickel cleaves only Michael adducts of HNE to cysteine via a thioether bond (HNE-SP), results from this study demonstrate that our GCMS method also detects with precision picomoles of HNE adducts via nitrogen residues (HNE-NP). Specifically, evidence was obtained using various study models, including polyamino acids consisting of cysteine, lysine, and histidine and a biologically relevant molecule, albumin. Furthermore, we show that dinitrophenylhydrazine treatment before Raney nickel treatment can be used to discriminate and quantify the various HNE-P molecular species in plasma and blood samples from normal rats, which range between 0.15 and 3 pmol/mg protein or 10 to 600 nM. However, whereas HNE-SP predominated in whole blood, we detected HNE-NP only in plasma. We also identified another significant MS signal, which we attribute to protein-bound 1,4-dihydroxynonane (DHN-P) presumably formed from the enzymatic reduction of HNE-P. The distribution profile of all these species in plasma differed from that observed when physiologically relevant concentrations of albumin and HNE were incubated in vitro. Furthermore, interestingly, hypercholesterolemic rabbits showed higher plasma levels of HNE-NP, but not of DHN-P. Beyond documenting the presence of various types of HNE-P in circulating proteins, our results emphasize the importance of enzymatic mechanisms in situ as a factor determining their distribution in the various blood compartments under various conditions.
