Targeted LC–MS derivatization for aldehydes and carboxylic acids with a new derivatization agent 4-APEBA

Based on the template of a recently introduced derivatization reagent for aldehydes, 4-(2-(trimethylammonio)ethoxy)benzeneaminium dibromide (4-APC), a new derivatization agent was designed with additional features for the analysis and screening of biomarkers of lipid peroxidation. The new derivatization reagent, 4-(2-((4-bromophenethyl)dimethylammonio)ethoxy)benzenaminium dibromide (4-APEBA) contains a bromophenethyl group to incorporate an isotopic signature to the derivatives and to add additional fragmentation identifiers, collectively enhancing the abilities for detection and screening of unknown aldehydes. Derivatization can be achieved under mild conditions (pH 5.7, 10 °C). By changing the secondary reagent (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide instead of sodium cyanoborohydride), 4-APEBA is also applicable to the selective derivatization of carboxylic acids. Synthesis of the new label, exploration of the derivatization conditions, characterization of the fragmentation of the aldehyde and carboxylic acid derivatives in MS/MS, and preliminary applications of the labeling strategy for the analysis of aldehydes in urine and plasma are described

Low-Density Lipoprotein Has an Enormous Capacity To Bind (E)-4-Hydroxynon-2-enal (HNE): Detection and Characterization of Lysyl and Histidyl Adducts Containing Multiple Molecules of HNE

(E)-4-Hydroxynon-2-enal (HNE), an electrophilic bifunctional cytotoxic lipid peroxidation product, forms covalent adducts with nucleophilic side chains of amino acid residues. HNE-derived adducts have been implicated in many pathophysiological processes including atherosclerosis, diabetes, and Alzheimer’s disease. Tritium- and deuterium-labeled HNE (d4-HNE) were used orthogonally to study adduction with proteins and individual nucleophilic groups of histidyl, lysyl, and cysteine residues. Using tritium-labeled HNE, we detected the binding of 486 molecules of HNE per low-density lipoprotein (LDL) particle, significantly more than the total number of all reactive nucleophiles in the LDL particle. This suggests the formation of adducts that incorporate multiple molecules of HNE with some nucleophilic amino acid side chains. We also found that the reaction of a 1:1 mixture of d4-HNE and d0-HNE with N-acetylhistidine, N-acetyl-Gly-Lys-OMe, or N-acetyl cysteine generates 1:1, 2:1, and 3:1 adducts, which exhibit unique mass spectral signatures that aid in structural characterization. A domino-like reaction of initial 1:1 HNE Michael adducts of histidyl or lysyl nucleophiles with multiple additional HNE molecules forms 2:1 and 3:1 adducts that were structurally characterized by tandem mass spectrometry

Les biomarqueurs de la peroxydation lipidique

Different biomarkers related to lipid peroxidation are available to assess an oxidative stress. They are more or less specific and sensitive. For instance malondialdehyde (MDA) or conjugated diene are easily measured in different matrix. Their measurements provide only a rough indication assessing an oxidative stress. But sometimes information is wrong due to artefacts. Some markers such as isoprostanes are specific of arachidonic acid only, and they require expensive equipment such as GC-MS/MS. They are mainly dedicated to clinical investigation. We present here new markers of oxidative stress which allow to discriminate between the oxidation of polyunsaturated fatty acid families. They are 4-hydroxynonenal (4-HNE) and 4-hydroxy-hexenal (4-HHE) which represent the peroxidation of the n-6 and n-3 polyunsaturated fatty acids, respectively and 4-hydroxy-dodecadienal (4-HDDE) which derives from the breakdown of 12-HpETE and mainly reflects the 12 lipoxygenation of arachidonic acid. Those markers can be easily measured with a high sensitivity by GC-MS in plasma. Their urinary carboxylic oxidized end-products, namely 4-HNA, 4-HHA and 4-HDDA may also be measured by GC-MS. The choice between these different biomarkers is not easy. It depends on which information is required