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

A new liquid chromatography-tandem mass spectrometric (LC-MS/MS) approach, based on the precursor ion scanning technique using a triple-stage quadrupole, has been developed to detect free and protein-bound histidine (His) residues modified by reactive carbonyl species (RCS) generated by lipid peroxidation. This approach has been applied to urines from Zucker obese rats, a nondiabetic animal model characterized by obesity and hyperlipidemia, where RCS formation plays a key role in the development of renal and cardiac dysfunction. The immonium ion of His at m/z 110 was used as a specific product ion of His-containing peptides to generate precursor ion spectra, followed by MS2 acquisitions of each precursor ion of interest for structural characterization. By this approach, three novel adducts, which are excreted in free form only, have been identified, two of them originating from the conjugation of 4-hydroxy-trans-2-nonenal (HNE) to His, followed by reduction/oxidation of the aldehyde: His-1,4-dihydroxynonane (His-DHN), His-4-hydroxynonanoic acid (His-HNA), and carnosine-HNE, this last recognized in previous in vitro studies as a new potential biomarker of carbonyl stress. No free His-HNE was found in urines, which was detected only in protein hydrolysates. The same LC-MS/MS method, working in multiple reaction monitoring (MRM) mode, has been developed, validated, and applied to quantitatively profile in Zucker urines both conventional (1,4-dihydroxynonane mercapturic acid, DHN-MA) and the newly identified adducts, except His-HNA. The analytes were separated on a C12 reversed-phase column by gradient elution from 100% A (water containing 5 mM nonafluoropentanoic acid) to 80% B (acetonitrile) in 24 min at a flow rate of 0.2 mL/min and analyzed for quantification in MRM mode by applying the following precursor-to-product ion transitions m/z 322.2 --> 164.1 + 130.1 (DHN-MA), m/z 314.7 --> 268.2 + 110.1 (His-DHN), m/z 312.2 --> 110.1 + 156.0 (His-HNE), m/z 383.1 --> 266.2 + 110.1 (CAR-HNE), m/z 319.2 --> 301.6 + 156.5 (H-Tyr-His-OH, internal standard). Precision and accuracy data, as well as the lower limits of quantification in urine, were highly satisfactory (from 0.01 nmol/mL for CAR-HNE, His-DHN, His-HNE, to 0.075 nmol/mL for DHN-MA). The method, applied to evaluate for the first time the advanced lipoxidation end products profile in urine from obese Zucker rats, an animal model for the metabolic syndrome, has proved to be suitable and sensitive enough for testing in vivo the carbonyl quenching ability of newly developed RCS sequestering agents