POST TRANSLATIONAL OXIDATIVE MODIFICATIONS OF ALBUMIN AS POTENTIAL BIOMARKERS FOR THE EARLY DIAGNOSIS OF CARDIOVASCULAR DISEASES AND AS A TOOL TO EVALUATE THE EFFICACY OF REACTIVE CARBONYL SPECIES SEQUESTERING AGENTS

4-hydroxy-trans-2-nonenal (HNE) is the most abundant and toxic alfa,beta-unsaturated aldehyde generated through the beta-cleavage of hydroperoxides from omega-6 PUFAs, which plays a key role in a variety of pathophysiological processes, including Alzheimer’s and Parkinson's diseases, liver fibrosis, glomerusclerosis, chronic obstructive bronco-pulmonary diseases and atherosclerosis. HNE reactivity is because of the conjugation of the double bond with the aldehyde function, which, by tautomeric equilibrium, makes the C-3 carbon a strong electrophilic center, able to react with cellular nucleophiles and in particular with the nucleophilic sites of protein (through a Michael addition mechanism), such as the sulfhydryl groups of cysteines, the imidazole moiety of histidines and the epsilon-amino group of lysine residues. In plasma, HNE has been found to modify LDL proteins. However LDL proteins probably represents only a minor protein target of HNE, since only 15% of the total plasma HNE-pyrrole immunoreactivity is removed by immunoprecipitation of apo B, thus to suggest that most of the oxidative metabolites generated by lipid oxidation bind to plasma proteins other than LDL proteins. Since albumin is present at high concentration in serum and is characterized by several nucleophilic and accessible residues, it should represents a favourable target protein but no detailed studies on the reaction between human serum albumin (HSA) and HNE have been up to now reported, nor on the chemical characterization of the protein adduct. Hence, aim of the thesis was to give evidences that HNE forms covalent addition products with HSA and to identify and characterize by electrospray ionization mass spectrometry (ESI-MS) and LC-ESI-MS/MS experiments the protein modification sites. As a first step, we found by HPLC analysis that HNE is rapidly quenched by HSA because of the covalent adduction to the different accessible nucleophilic residues of the protein, as demonstrated by electrospray ionization mass spectrometry (ESI-MS) direct infusion experiments (one to nine HNE adducts, depending on the molar ratio used, from 1 : 0.25 to 1 : 5 HSA : HNE). An LC-ESI-MS/MS approach was then applied to enzymatically digested HNE-modified albumin, which permitted the identification of 11 different HNE adducts, 8 Michael adducts (MA) and 3 Schiff bases (SB), involving nine nucleophilic sites, namely: His67 (MA), His146 (MA), His242 (MA), His288 (MA), His510 (MA), Lys 195 (SB), Lys 199 (MA, SB), Lys525 (MA, SB) and Cys34 (MA). The most reactive HNE-adduction site was found to be Cys34 (MA) followed by Lys199, which primarily reacts through the formation of a Schiff base, and His146, giving the corresponding HNE Michael adduct. Albumin modified by HNE can be a promising biomarker of circulating HNE and a systemic oxidative stress index. In fact, aldehyde-protein adducts have a longer half-life than the corresponding free aldehydes, and the relative long half-life of albumin in human subjects (15-19 days) in respect to other plasma proteins, makes it an ideal biomarker of oxidative stress. In the present thesis we found that HNE-adducted peptides and in particular the HNE derivatives of Cys34, His146 and Lys199 containing peptides derived by proteolytic-enzymes digestion could be suitable tags of HNE-adducted albumin.