HNE Michael adducts to histidine and histidine-containing peptides as new biomarkers of lipid-derived carbonyl stress in urines: LC-MS/MS profiling in Zucker obese rats

Reactive carbonyl species (RCS) generated by lipid peroxidation, such as 4-hydroxy-trans-2-nonenal (HNE), acrolein (ACR), malondialdehyde (MDA) leading to covalently modified peptides and proteins, are known to be involved in the pathogenesis of several cardiovascular (atherosclerosis, cerebral or heart ischemia-reperfusion injury) and neurodegenerative (Alzheimer’s disease, Parkinson’s disease) disease, as well as in diabetes and cancer [1]. The mainly used in vivo biomarkers for carbonylation seem to be Cys and Lys residues, while little attention has been devoted to covalently modified His as a possible diagnostic tool. The first aim of this work was to develop a LC-MS/MS approach, based on the precursor ion scanning mode, to detect all free and protein-bound His species modified by RCS. This technique, being a valuable tool for the rapid confirmation of targeted compounds or for the non-targeted detection of compounds bearing a common moiety, has never been applied to screen a biological matrix for the presence of covalently modified peptides. We used the immonium ion of His (m/z 110.2) as a well known and specific fragment ion of His-containing peptides [2] to generate precursor ion spectra in order to screen urines from Zucker obese rats for the presence of covalently modified His residues. The Zucker rat has been chosen as a non-diabetic animal model characterized by obesity and hyperlipidemia, where carbonylation plays a key role in the development of renal and cardiac dysfunction [3]. This scan mode was followed by an enhanced resolution experiment for the ions of interest and then by MS2 acquisitions for structure assignement. The combined approach allowed identifying for the first time in urine from obese Zucker rats three novel adducts, originating from the conjugation of HNE to His: His-DHN (free form), arising from the subsequent enzymatic reduction of the aldehydes; His-HNA (free form), by conjugation of HNE followed by enzymatic oxidation of the aldehydes; His-HNE (protein-bound). Furthermore, the results indicate excretion of the Michael adduct of HNE with the endogenous dipeptide Carnosine (β-alanyl-L-histidine, CAR), previously identified in rat skeletal muscle as a marker of carbonylation, but never identified in urines [2]. The second aim of this work was to develop and validate a sensitive and selective LC-MS/MS approach in multiple reaction monitoring (MRM) mode for the quantitative determination in urine from Zucker rats of both conventional (DHN-MA, the final urinary product of HNE conjugated to glutathione), and the newly identified adducts to His. Separations (reverse phase) were performed by gradient elution from 100% water containing 5 mM nonafluoropentanoic acid (A) to 80% acetonitrile (B) in 24 min at a flow rate of 0.2 ml/min and analyzed by mass spectrometry (ESI) in positive ion mode. The results of this study show that the adducts excretion, being negligible in young control rats, becomes evident in old control animals, and significantly increased in the pathological model (Zucker rats). This confirms that adduct formation takes place in physiological conditions and is strictly associated with aging, and indicate that the Zucker obese rat represents an appropriate animal model of metabolic syndrome for testing in vivo the carbonyl quenching ability of newly developed sequestering agents, by measuring the urinary levels of the highly stable biomarkers considered in this study.