4-hydroxy-2-nonenal and sodium-borohydride

The octapeptide angiotensin II (Ang II; Asp(1)-Arg(2)-Val(3)-Tyr(4)-Ile(5)-His(6)-Pro(7)-Phe(8)) is the primary active hormone of the renin/angiotensin system (RAS) and has been implicated in various cardiovascular diseases. Numerous structure-activity relationship studies have identified Asp(1), Arg(2), and His(6) of Ang II to be critical for its biological activity and receptor binding. From the reactions of Ang II with lipid peroxidation-derived aldehydes, 4-oxo-2(E)-nonenal (ONE) or 4-hydroxy-2(E)-nonenal (HNE), we have identified the major modifications to the N-terminus, Asp(1), Arg(2), and His(6) of Ang II by liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption ionization-time-of-flight/MS (MALDI-TOF/MS). The identities of ONE- and HNE-modified Ang II were confirmed by tandem mass spectrometry (MS/MS) and postsource decay (PSD)-TOF/MS before and after the reaction with sodium borohydride. In the reaction with ONE, a pyruvamide-Ang II that formed via oxidative decarboxylation of N-terminal Asp was detected as the most abundant product after 48 h of incubation. It was followed by Arg-modified [Arg(2)(ONE-H(2)O)]-Ang II and the N-terminal-modified 4-ketoamide form of [N-ONE]-Ang II. The Michael addition products of [His(6)(HNE)]-Ang II were the most abundant products in the beginning of the reaction with HNE, followed by the dehydrated Michael addition products of [His(6)(HNE-H(2)O)]-Ang II. [His(6)(HNE)]-Ang II was dehydrated to [His(6)(HNE-H(2)O)]-Ang II during the prolonged incubation, and [His(6)(HNE-H(2)O)]-Ang II became the major products after 7 days. The model reactions of N(α)-tert-butoxycarbonyl (tBoc)-Arg with ONE and tBoc-His with HNE were performed and compared with the Ang II reaction. tBoc-Arg readily reacted with ONE to produce a compound analogous to [Arg(2)(ONE-H(2)O)]-Ang II, which confirmed Arg as one of the important target nucleophiles of ONE. However, tBoc-His exclusively formed a Michael addition product upon the reaction with HNE. The unexpected formation of [His(6)(HNE-H(2)O)]-Ang II can be explained by the proximity of His(6) to C-terminal carboxylate in the specific conformation of Ang II, which facilitates the dehydration of Michael addition products. Therefore, our results suggest a possible discrepancy in the adduction chemistry of ONE and HNE for model amino acids and endogenous bioactive peptides, which is governed by the microenvironment of peptides, such as the specific amino acid sequ

Topics: Aldehydes; Amino Acid Sequence; Angiotensin II; Borohydrides; Chromatography, High Pressure Liquid; Lipid Peroxidation; Oligopeptides; Oxidation-Reduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Time Factors

Keratinocytes are potential targets of lipid peroxidation products (alpha,beta-unsaturated aldehydes) generated in the skin following UV exposure, among which the most abundant and toxic product is 4-hydroxy-trans-2,3-nonenal (HNE). The aim of this study was to investigate the ability of keratinocytes (NCTC2544 cell lines) to detoxify HNE, through characterization of metabolites, until now never demonstrated, using a combined analytical approach (liquid chromatography (LC) and liquid chromatography/mass spectrometry (LC/MS)). Incubation of cells with HNE (up to 200 micro M) was performed in order to evaluate the ability of the cells to detoxify this toxic aldehyde, and indicated that the cell viability was maintained under these conditions. LC analysis of the extracellular media from keratinocytes incubated with 100 micro M HNE shows a time-dependent decrease of HNE, disappearance from the medium within 2 h and concomitant formation of two unconjugated (phase I) metabolites, 4-hydroxy-2-nonenoic acid (HNA) and 1,4-dihydroxy-2-nonene (DHN), which were both identified and quantified by LC and accounted for 48.8 +/- 4.6% of the HNE dose. Four additional metabolites were identified in the extracellular medium by reversed-phase LC coupled with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) with positive and negative ion detection as Michael adducts (phase II metabolites), arising by the addition of the nucleophilic sulfur of glutathione (GSH) to the electrophilic C-3 of HNE, followed by oxidation-reduction enzymatic processes. The GSH-HNE conjugates were (a) S-(4-hydroxynonanal-3-yl)glutathione, (b) S-(1,4-dihydroxy-nonane-3-yl)glutathione, (c) S-(4-oxononanal-3-yl)glutathione and (d) S-(4-oxo-nonan-1-ol-3-yl)glutathione, and accounted for 52.3 +/- 5.8% of the HNE dose (35 nmol mg(-1) protein), as estimated indirectly by measuring the extent of cellular GSH consumption (18.7 +/- 1.8 nmol mg(-1) protein). The time course of HNE biotransformation was then determined by monitoring the formation of metabolites inside and outside the cell at different times after HNE addition (5-120 min). A time-dependent and almost linear formation inside the cell was observed for all the metabolites (plateau after 15 min of incubation), followed by a rapid decay and a concomitant increase in the extracellular medium (plateau of formation after 60 min). This confirms that HNE diffuses into the cell where is totally metabolized through phase I and phase II reaction

Topics: Aldehydes; Borohydrides; Cell Line; Chromatography, Liquid; Humans; Inactivation, Metabolic; Keratinocytes; Kinetics; Molecular Structure; Spectrometry, Mass, Electrospray Ionization

The toxicity of the beta-amyloid (Abeta) peptide of Alzheimer's disease may relate to its polymerisation state (i.e. fibril content). We have shown previously that plasma lipoproteins, particularly when oxidised, greatly enhance Abeta polymerisation. In the present study the nature of the interactions between both native and oxidised lipoproteins and Abeta1-40 was investigated employing various chemical treatments. The addition of ascorbic acid or the vitamin E analogue, trolox, to lipoprotein/Abeta coincubations failed to inhibit Abeta fibrillogenesis, as did the treatment of lipoproteins with the aldehyde reductant, sodium borohydride. The putative lipid peroxide-derived aldehyde scavenger, aminoguanidine, however, inhibited Abeta-oxidised lipoprotein-potentiated polymerisation, but in a manner consistent with an antioxidant action for the drug. Lipoprotein treatment with the reactive aldehyde 4-hydroxy-2-trans-nonenal enhanced Abeta polymerisation in a concentration-dependent fashion. Incubation of Abeta with lipoprotein fractions from which the apoprotein components had been removed resulted in extents of polymerisation comparable to those observed with Abeta alone. These data indicate that the apoprotein components of plasma lipoproteins play a key role in promoting Abeta polymerisation, possibly via interactions with aldehydes.

Topics: Aldehydes; Alzheimer Disease; Amyloid beta-Peptides; Antioxidants; Apolipoproteins; Ascorbic Acid; Biopolymers; Borohydrides; Chromans; Guanidines; Humans; Kinetics; Lipoproteins; Oxidation-Reduction; Peptide Fragments