9-Oxononanoic Acid and Its Lysine Schiff Base Adduct as a Novel Lipation Product in Peanuts.

9-Oxononanoic acid (9-ONA) was quantitated in peanuts roasted at 170 °C by GC-MS (EI). After roasting peanuts for 40 min, 9-ONA decreased from 1010 µmol/kg protein in the unheated sample to 722 µmol/kg protein, most likely due to modifications of nucleophilic side chains of protein-bound amino acids (lipation). After heating Nα-acetyl-l-lysine and 9-ONA in model experiments, a Schiff base in its reduced form, namely, Nε-carboxyoctyl-acetyl lysine, as well as two isomeric pyridinium derivatives, namely, dicarboxyhexylcarboxyheptylpyridinium-acetyl lysine 1 and 2, were tentatively identified by HPLC-ESI-MS/MS. Based on the identified lipation products of 9-ONA, it can be assumed that lipation reactions represent a mirror-image reaction. For quantitation of Nε-carboxyoctyllysine (COL) in roasted peanuts by means of HPLC-ESI-MS/MS, samples were reduced with sodium borohydride and acid hydrolyzed. For the first time, COL was quantitated after reduction in roasted peanuts. Furthermore, after prolonged roasting of peanuts for 40 min, COL decreased from 139.8 to 22.5 µmol/kg protein, which provides initial evidence for lipation of nucleophilic side chains of protein-bound amino acids by glycerol-bound oxidized fatty acids (GOFAs, e.g., 9-ONA) with the formation of neo-lipoproteins.

Human monocyte-derived type 1 and 2 macrophages recognize Ara h 1, a major peanut allergen, by different mechanisms.

Evidence has suggested that major peanut allergen Ara h 1 activates dendritic cells (DCs) via interaction with DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin), a C-type lectin receptor, and contributes to development of peanut allergy. Since macrophages, as well as DCs, play a crucial role in innate immunity, we investigated whether natural Ara h 1 (nAra h 1) activates two different subsets of macrophages, human monocyte derived macrophage type 1 (hMDM1: pro-inflammatory model) and type 2 (hMDM2: anti-inflammatory model). hMDM1 and hMDM2 predominantly produced pro-inflammatory cytokines (IL-6 and TNF-α) and an anti-inflammatory cytokine (IL-10) in response to nAra h 1, respectively. hMDM2 took up nAra h 1 and expressed DC-SIGN at higher levels than hMDM1. However, small interfering RNA knockdown of DC-SIGN did not suppress nAra h 1 uptake and nAra h 1-mediated cytokine production in hMDM2. Inhibitors of scavenger receptor class A type I (SR-AI) suppressed the response of hMDM2, but not of hMDM1, suggesting that SR-AI is a major receptor in hMDM2 for nAra h 1 recognition and internalization. nAra h 1 appears to exert stimulatory capacity on DC and macrophages via different receptors. This study advances our understanding how a major peanut allergen interacts with innate immunity.

Identification and Quantitation of the Lipation Product 2-Amino-6-(3-methylpyridin-1-ium-1-yl)hexanoic Acid (MP-Lysine) in Peanuts.

The lipid peroxidation product acrolein was semiquantitated by GC-MS (EI) in unheated and heated peanut oil, respectively, representing a model system for peanut roasting. Depending on the heating time, acrolein levels significantly increased from 0.2 to 10.7 mg/kg oil. As a result of heating N(α)-acetyl-l-lysine and acrolein, the pyridinium derivative 2-acetamido-6-(3-methylpyridin-1-ium-1-yl)hexanoic acid (MP-acetyl lysine) was identified. In addition, the lysine derivative 2-amino-6-[5-(hydroxymethyl)-3,6-dihydro-2H-pyridin-1-yl]hexanoic acid was identified after reduction and hydrolysis. After preparation of 2-amino-6-(3-methylpyridin-1-ium-1-yl)hexanoic acid (MP-lysine) as reference material, its amounts were quantitated in acrolein-modified peanut proteins by HPLC-ESI-MS/MS after acid hydrolysis, showing that at low acrolein concentrations, the modification of lysine could be entirely explained by the formation of MP-lysine. Furthermore, for the first time, MP-lysine was quantitated in peanut samples in amounts up to 10.2 mg/kg, showing an increase depending on the roasting time. Thus, MP-lysine might represent a marker to evaluate the extent of food protein lipation by acrolein.

4-Hydroxy-2-nonenal (4-HNE) and Its Lipation Product 2-Pentylpyrrole Lysine (2-PPL) in Peanuts.

After synthesis of a deuterated 4-hydroxy-2-nonenal (4-HNE) standard, the formation of 4-HNE during heating of peanut oil and whole peanuts, respectively, was measured by GC-MS. Whereas a significant increase in 4-HNE levels was observed for peanut oil, the amount of 4-HNE decreased when whole peanuts were roasted due to lipation reactions with amino acid side chains of the proteins. The ε-amino group of lysine was identified as the favored reaction partner of 4-HNE. After heating N(α)-acetyl-l-lysine and 4-HNE, a Schiff base, a novel pyridinium derivative, a 2-pentylpyrrol derivative and, following reduction and hydrolysis, a reduced, cyclized Michael adduct were identified. 2-Amino-6-(2-pentyl-1H-pyrrol-1-yl)hexanoic acid (2-PPL) was synthesized and quantitated in peanut proteins, which had been incubated with various amounts of 4-HNE by HPLC-ESI-MS/MS after enzymatic hydrolysis. At low 4-HNE concentrations the modification of lysine could be entirely explained by the formation of 2-PPL. Additionally, 2-PPL was quantified for the first time in peanut samples, and an increase depending on the roasting time was observed. 2-PPL represents a suitable marker to evaluate the extent of food protein lipation by 4-HNE.

Studies on the reaction of trans-2-heptenal with peanut proteins.

Hexanal, 2-heptenal, and nonanal were identified as relevant reaction products formed in the course of the lipid peroxidation of heated peanut oil. For the identification of potential amino acid side chain adducts, kinetic studies between N(α)-benzoylglycyl-l-lysine as a model for protein-bound lysine and trans-2-heptenal were performed, showing a strong decrease of the lysine-derivative whereupon the loss of trans-2-heptenal was moderate. Following acid hydrolysis of the incubation mixture of N(α)-acetyl-l-lysine and trans-2-heptenal, two UV-active major lipation products were observed, isolated and identified as isomeric pyridinium-derivatives, namely (Z)- and (E)-1-(5-amino-5-carboxypentyl)-4-butyl-3-(pent-1-en-1-yl)pyridin-1-ium (cis- and trans-BPP-lysine). After heating of a native peanut protein extract with trans-2-heptenal, both derivatives were quantitated by LC-ESI-MS/MS after acid hydrolysis and the modification of lysine was measured by amino acid analysis. At low, "food-relevant", concentrations of trans-2-heptenal, up to 80% of the lysine modification could be explained by the formation of cis- and trans-BPP-lysine, showing that these two lipation derivatives represent good markers for a protein modification by the lipid peroxidation product trans-2-heptenal.