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4-Hydroxynonenal (HNE), an electrophilic end-product deriving from lipid peroxidation, undergoes a heterogeneous set of biotransformations including enzymatic and non-enzymatic reactions. The former mostly involve red-ox reactions on the HNE oxygenated functions (phase I metabolism) and GSH conjugations (phase II) while the latter are due to the HNE capacity to spontaneously condense with nucleophilic sites within endogenous molecules such as proteins, nucleic acids and phospholipids. The overall metabolic fate of HNE has recently attracted great interest not only because it clearly determines the HNE disposal, but especially because the generated metabolites and adducts are not inactive molecules (as initially believed) but show biological activities even more pronounced than those of the parent compound as exemplified by potent pro-inflammatory stimulus induced by GSH conjugates. Similarly, several studies revealed that the non-enzymatic reactions, initially considered as damaging processes randomly involving all endogenous nucleophilic reactants, are in fact quite selective in terms of both reactivity of the nucleophilic sites and stability of the generated adducts. Even though many formed adducts retain the expected toxic consequences, some adducts exhibit well-defined beneficial roles as documented by the protective effects of sublethal concentrations of HNE against toxic concentrations of HNE. Clearly, future investigations are required to gain a more detailed understanding of the metabolic fate of HNE as well as to identify novel targets involved in the biological activity of the HNE metabolites. These studies are and will be permitted by the continuous progress in the analytical methods for the identification and quantitation of novel HNE metabolites as well as for proteomic analyses able to offer a comprehensive picture of the HNE-induced adducted targets. On these grounds, the present review will focus on the major enzymatic and non-enzymatic HNE biotransformations discussing both the molecular mechanisms involved and the biological effects elicited. The review will also describe the most important analytical enhancements that have permitted the here discussed advancements in our understanding of the HNE metabolic fate and which will permit in a near future an even better knowledge of this enigmatic molecule., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Carnosine aryl derivatives as sequestering agents of RCS: Reactive carbonyl species (RCS) are cytotoxic mediators representing a novel drug target, as they are presumed to play a pathogenic role in several diseases. Carnosine is a selective RCS-sequestering agent, but is rapidly hydrolyzed by serum carnosinase. Herein we describe the in silico design, synthesis, and evaluation of a set of carnosine aryl derivatives.Reactive carbonyl species (RCS) are important cytotoxic mediators generated by lipid oxidation of polyunsaturated fatty acids (PUFAs) and represent a novel drug target, as they are presumed to play a pathogenic role in several diseases. L-Carnosine (L-CAR, beta-alanyl-L-histidine) is a specific detoxifying agent of RCS, but is rapidly hydrolyzed in human serum by carnosinase, a specific dipeptidase. Herein we describe the in silico design, synthesis, and biological evaluation of carnosine derivatives that are resistant to carnosinase and that have increased quenching efficacy. Stability against carnosinase-mediated turnover was achieved by isomerization of the histidine residue, leading to D-carnosine (D-CAR, beta-alanyl-D-histidine), which maintains the same quenching activity of L-carnosine. A molecular modeling approach was then used to design derivatives characterized by an increased quenching efficacy. The most promising candidates were synthesized, and their stability and quenching activity were evaluated. This study describes a set of aryl derivatives that are characterized by high stability in human plasma and a quenching activity toward 4-hydroxy-trans-2-nonenal (HNE), chosen as a model of RCS, up to threefold greater than D-carnosine.

Most of the assays for detection of carbonylated proteins, the most general and widely used marker of severe protein oxidation, involve derivatization of the carbonyl group with 2,4-dinitrophenylhydrazine (DNPH), which leads to formation of a stable dinitrophenyl hydrazone product. Here, by using a Cys-containing model peptide and high-resolution mass spectrometry, we demonstrate that DNPH is not exclusively selective for carbonyl groups, because it also reacts with sulfenic acids, forming a DNPH adduct, through the acid-catalyzed formation of a thioaldehyde intermediate that is further converted to an aldehyde. beta-Mercaptoethanol prevents the formation of the DNPH derivative because it reacts with the oxidized Cys residue, forming the corresponding disulfide.

The aim of this work was to investigate in vitro the quenching activity of green tea polyphenols against alpha,beta-unsaturated aldehyde, using 4-hydroxy-nonenal (HNE) as prototype and HPLC-ESI-MS/MS techniques. HNE is the most abundant and genotoxic product of oxidation of dietary polyunsaturated fatty acids, and is believed to be involved in the early stage of colorectal carcinogenesis on account of its genotoxic potential. Both epigallocatechin gallate (EGCG, 1.0-3.5mM), the main constituent of green tea polyphenols, and a green tea aqueous extract are able to quench HNE (50 microM) in colorectal physiomimetic conditions (10mM phosphate buffer, pH 8.0, 37 degrees C), giving rise to the formation of six diastereomeric covalent adducts at the ring A of EGCG, as indicated by their ESI-MS/MS fragmentation pathways. The specificity of the adduction positions was explained by (1)H NMR experiments. HNE quenching is pH-dependent and maximum at pH 8.0. ESI-MS analysis showed no formation of 4-hydroxy-2,3-epoxy-nonanal, or adduction of the epoxide to EGCG. This implies that too little hydrogen peroxide (1mM, 24h incubation, FOX-2 method) develops from auto-oxidation of EGCG in our aerobic experimental conditions to oxidize HNE to its corresponding epoxide, so this mechanism is not responsible for the compound's disappearance. EGCG and green tea extract also quenched acrolein, another genotoxic alpha,beta-unsaturated aldehyde, giving one predominant adduct and minor isobaric species, probably due the adduction of acrolein at different positions of the EGCG ring A. These results suggest that EGCG and green tea extract, beside the proposed mechanisms of chemoprevention that target multiple cell-signaling pathways that control cell proliferation and apoptosis in cancer cells, can also prevent protein carbonylation in the tumor tissue environment, depending on the pH of the medium surrounding the tissue, the type of tumor, the stage of dysregulation of lipid peroxidation and, finally, the stage of carcinoma development.

The aim of this work was to study the metabolic fate of 4-hydroxy- trans-2-nonenal (HNE) in human plasma, which represents the main vascular site of reactive carbonyl species (RCS) formation and where the main pro-atherogenic target proteins are formed. When HNE was spiked in human plasma, it rapidly disappeared (within 40 s) and no phase I metabolites were detected, suggesting that the main fate of HNE is due to an adduction mechanism. HNE consumption was then monitored in two plasma fractions: low molecular weight plasma protein fractions (<10 kDa; LMWF) and high molecular weight plasma protein fractions (>10 kDa; HMWF). HNE was almost stable in LMWF, while in HMWF it was consumed by almost 70% within 5 min. Proteomics identified albumin (HSA) as the main protein target, as further confirmed by a significantly reduced HNE quenching of dealbuminated plasma. LC-ESI-MS/MS analysis identified Cys34 and Lys199 as the most reactive adduction sites of HSA, through the formation of a Michael and Schiff base adducts, respectively. The rate constant of HNE trapping by albumin was 50.61 +/- 1.89 M (-1) s (-1) and that of Cys34 (29.37 M (-1) s (-1)) was 1 order of magnitude higher with respect to that of GSH (3.81 +/- 0.17 M (-1) s (-1)), as explained by molecular modeling studies. In conclusion, we suggest that albumin, through nucleophilic residues, and in particular Cys34, can act as an endogenous detoxifying agent of circulating RCS.

Histidine-containing oligopeptides are currently studied as detoxifying agents against cytotoxic alpha,beta-unsaturated aldehydes (prototype: 4-hydroxy-2-nonenal, HNE), electrophilic end products formed by decomposition of omega-6 polyunsaturated fatty acids, associated with severe pathologies such as diabetes, nephropathy, retinopathy, and neurodegenerative diseases. This study evaluated the quenching reaction against HNE of the endogenous tripeptide l-glycyl- l-histidyl- l-lysine (GHK), an oligopeptide discovered to be a growth-modulating factor and also a strong activator of wound healing. We first evaluated the HNE consumption (50 microM, HPLC-UVDAD method) in the presence of GHK (1 mM) in physiomimetic conditions (phosphate buffer, pH 7.4) and confirmed GHK/HNE adduct formation by mass spectrometric analysis (ESI-MS/MS) and (1)H NMR analyses. These results indicated that GHK was an effective quencher of HNE, although significantly less potent than the reference compound carnosine, and that HNE modulation by GHK can contribute to the satisfactory outcome of the wound-healing process. In the second part of the study, we investigated the quenching reaction between GHK and HNE, in parallel to carnosine, using (1)H NMR and computational analyses. At a mechanistic level, this explained the different reactivity of the two peptides: (i) The greater stability of the macrocyclic intermediate HNE/carnosine was compared to HNE/GHK. (ii) GHK in solution has a quasi-folded conformation due to the interaction of four intramolecular hydrogen bonds, three of which need to be broken for the transition state to form (energy barrier, approximately 20 kcal/mol). By contrast, carnosine, with an extended conformation and only one hydrogen bond, requires less energy to reach the transition state ( approximately 7 kcal/mol). (iii) The different stereoelectronic features of the transition state lead to the intramolecular Michael reaction, that is, the favorable superimposition of carnosine highest occupied molecular orbital and the HNE lowest unoccupied molecular orbital, in relation to the unfavorable orbital configuration of GHK. The overall findings provide interesting and useful insights into the mechanisms of interaction of both GHK and carnosine with HNE and illustrate the utility of computational studies for defining the (optimal) chemical and structural parameters for an optimal quenching of alpha,beta-unsaturated aldehydes.

Several pieces of evidence indicate that albumin modified by HNE is a promising biomarker of systemic oxidative stress and that HNE-modified albumin may contribute to the immune reactions triggered by lipid peroxidation-derived antigens. In this study, we found by HPLC analysis that HNE is rapidly quenched by human serum albumin (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. These albumin modifications are suitable tags of HNE-adducted albumin and could be useful biomarkers of oxidative and carbonylation damage in humans., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

Sugar- and lipid-derived aldehydes are reactive carbonyl species (RCS) frequently used as surrogate markers of oxidative stress in obesity. A pathogenic role for RCS in metabolic diseases of obesity remains controversial, however, partly because of their highly diffuse and broad reactivity and the lack of specific RCS-scavenging therapies. Naturally occurring histidine dipeptides (e.g., anserine and carnosine) show RCS reactivity, but their therapeutic potential in humans is limited by serum carnosinases. Here, we present the rational design, characterization, and pharmacological evaluation of carnosinol, i.e., (2S)-2-(3-amino propanoylamino)-3-(1H-imidazol-5-yl)propanol, a derivative of carnosine with high oral bioavailability that is resistant to carnosinases. Carnosinol displayed a suitable ADMET (absorption, distribution, metabolism, excretion, and toxicity) profile and was determined to have the greatest potency and selectivity toward [alpha],[beta]-unsaturated aldehydes (e.g., 4-hydroxynonenal, HNE, ACR) among all others reported thus far. In rodent models of diet-induced obesity and metabolic syndrome, carnosinol dose-dependently attenuated HNE adduct formation in liver and skeletal muscle, while simultaneously mitigating inflammation, dyslipidemia, insulin resistance, and steatohepatitis. These improvements in metabolic parameters with carnosinol were not due to changes in energy expenditure, physical activity, adiposity, or body weight. Collectively, our findings illustrate a pathogenic role for RCS in obesity-related metabolic disorders and provide validation for a promising new class of carbonyl-scavenging therapeutic compounds rationally derived from carnosine., IntroductionOvernutrition from fatty acids and complex carbohydrates is known to cause oxidative stress from multiple enzymatic and nonenzymatic sources due to the high caloric content and the prevalence of these [...]