4-Hydroxy-2-nonenal (4-HNE) - ACS Publications - American

May 6, 2015 - ABSTRACT: After synthesis of a deuterated 4-hydroxy-2-nonenal (4-HNE) standard, the formation of 4-HNE during heating of peanut oil and ...
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4‑Hydroxy-2-nonenal (4-HNE) and Its Lipation Product 2‑Pentylpyrrole Lysine (2-PPL) in Peanuts Martin Globisch, Diana Kaden, and Thomas Henle* Institute of Food Chemistry, Technische Universität Dresden, D-01062 Dresden, Germany S Supporting Information *

ABSTRACT: 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 Nα-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. KEYWORDS: peanut, peanut oil, lipid peroxidation, lipation, carbonyl protein reaction, 4-hydroxynonenal, 4-hydroxynon-2-enal, 4-hydroxy-2-nonenal, 4-HNE, HNE, 2-pentylpyrrole lysine, 2-PPL, 2-pentylpyrrole

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INTRODUCTION 4-Hydroxynon-2-enal (4-HNE) is a well-known secondary product of lipid peroxidation, which is mainly formed from ω-6 polyunsaturated fatty acids.1 The formation of secondary lipid peroxidation products during roasting of peanuts, which contain 14% linoleic acid,2 was shown,3 and the presence of 4-HNE in commercially available peanuts was recently published, ranging from 0.8 to 3.8 μmol/kg peanut.4 As a result of roasting peanuts, 30−40% of peptide-bound lysine was modified, but only 10% of this lysine modification could be explained by the formation of Maillard reaction products such as Nε-fructosyllysine, pyrraline, and Nε-carboxymethyllysine.5 The possibility for a reaction between secondary lipid peroxidation products and amino acid side chains in peanut proteins was recently shown for 2-heptenal, resulting in cis- and trans-BPP-lysine derivatives. For these protein−carbonyl reactions, the term “lipation” was suggested.6 Because of its ability to react easily with nucleophilic compounds, the formation of lipation products between 4-HNE and amino acid side chains of peanut proteins during roasting of peanuts is highly likely. Due to the three functional groups of 4-HNE, various reactions are possible. They include the formation of Michael addition products, which are able to undergo secondary reactions involving the carbonyl and the hydroxyl groups,7 resulting, for example, in stable, cyclic hemiacetals.8 After the formation of Schiff bases with primary amines, further reactions can lead to the formation of 2-pentylpyrrole residues,9 which was shown for bovine serum albumin incubated with 4HNE under physiological conditions.10 Using 13C NMR spectroscopy, 20% of the protein-bound 4-HNE was found as 2-pentylpyrrole,10 which was classified as an advanced lipoxidation endproduct (ALE).8,11 The incubation of RNase A © 2015 American Chemical Society

with 4-HNE in sodium phosphate buffer (pH 7.2) at room temperature for 5 h resulted in the formation of a 2-hydroxy-2pentyl-1,2-dihydropyrrol-3-one iminium fluorophore, accounting for protein cross-linking.12 The yield of the fluorophore was estimated to account for 1.8% of the lysine residues on the protein. Another possible cross-linking mechanism would be the simultaneous formation of a Michael adduct and a Schiff base at one molecule of 4-HNE.12,13 Using immunochemical methods, 4-HNE-pyrrole adducts were found to be significantly elevated in the plasma of patients with end-stage renal disease as well as in the plasma of atherosclerotic patients compared to that of healthy volunteers.14 Furthermore, 4-HNE−pyrrole adducts were detected in collagen fibers of an advanced atherosclerotic lesion. In another study, 4-HNE−lysine adducts in intact apo B and apo B fragments of atherosclerotic lesions were observed by Western blot analysis.15 Due to the fact that 4-HNE-pyrroles are stable advanced lipoxidation endproducts,8,11 their quantitative levels in human blood may be an indelible marker of 4-HNE generation in vivo.14 With regard to 4-HNE−protein adducts in food, only a few studies are available. Using an indirect ELISA, 4-HNE−protein adducts were detected in protein fractions of stored beef.16 However, the specific proteins and 4-HNE adducts that were detected by the antibodies were not known. In protein extracts prepared from defatted meals of raw and roasted peanuts, 4-HNE− protein adducts were detected by direct ELISA.17,18 In these studies, no significant differences in 4-HNE adduct levels Received: Revised: Accepted: Published: 5273

March 24, 2015 May 1, 2015 May 6, 2015 May 6, 2015 DOI: 10.1021/acs.jafc.5b01502 J. Agric. Food Chem. 2015, 63, 5273−5281

Article

Journal of Agricultural and Food Chemistry

lysine (2-PPL), 1H NMR spectra were recorded on a Bruker Avance 600 instrument (Rheinstetten, Germany) at 600.16 MHz. 4Hydroxynonenal dimethyl acetal (4-HNE-DMA) was measured on a Bruker DRX 500 instrument at 500.13 MHz. 13C NMR spectra were recorded on a Bruker DRX 500 instrument at 150.9 MHz, respectively. For fumaraldehyde dimethyl acetal and (E)-5,5-dideuterio-1,1dimethoxynon-2-en-4-ol dimethyl acetal (4-HNE-DMA-d2), 1H NMR and 13C NMR spectra were recorded on a Bruker Avance 400 instrument (Rheinstetten, Germany) at 400.13 MHz and at 100.61 MHz, respectively. For 4-HNE-DMA, 1,4-ND, 4-ON, fumaraldehyde dimethyl acetal, and 4-HNE-DMA-d2, deuterochloroform (CDCl3) and for 2-PPL tetradeuteromethanol (MeOH-d4) were used as solvent. All chemical shifts are given in parts per million (ppm) relative to the solvent signal serving as internal standard. The following 2D-NMR experiments were performed additionally for 4-HNE-DMA, 4-HNEDMA-d2 4-ON, 1,4-ND, and 2-PPL: correlation spectroscopy (COSY), nuclear Overhauser enhancement spectroscopy (NOESY), heteronuclear single-quantum coherence (HSQC), and heteronuclear multiple-bond correlation (HMBC). Elemental Analysis. Elemental analysis was performed using an EuroEA3000 (Eurovector, Milan, Italy) to quantitate the product content in the 2-PPL standard. Therefore, the analyzed percentage of nitrogen was compared to the calculated nitrogen percentage. The purity of 2-PPL is expressed in percent by weight. Acid and Enzymatic Hydrolysis. For acid hydrolysis, a ratio of 8 mg of protein per 1.0 mL of 6 M hydrochloric acid was used. Samples were hydrolyzed under nitrogen for 23 h at 110 °C. For enzymatic hydrolysis the method published elsewhere was used.6,20 Amino Acid Analysis. For measuring of the lysine modification rate and comparison of the release of amino acids after acid and enzymatic hydrolysis, amino acid analyses were performed using a SYKAM S4300 amino acid analyzer (Fürstenfeldbruck, Germany). Amino acids were separated by cationic ion exchange chromatography, derivatized by ninhydrin, and detected using a wavelength of 570 nm.21 Valine was taken as internal standard, and amino acids were expressed as valine equivalents. Synthesis of (E)-1,1-Dimethoxynon-2-en-4-ol (4-HNE-DMA). For 4-HNE-DMA synthesis, a method published elsewhere was adopted.22 Therefore, 25 mL of nitrogen-purged dichloromethane, 496 μL of 1-octen-3-ol, 1164 μL of acrolein dimethyl acetal, and 25.9 mg of a Hoveyda−Grubbs catalyst second generation were given in a 50 mL three-neck flask and stirred for 5 h at room temperature under nitrogen. After the addition of another 25.9 mg of catalyst, stirring was continued for 20 h at room temperature under nitrogen. After evaporation of dichloromethane, 4-HNE-DMA was purified by silica gel chromatography using diethyl ether/pentane (50:50, v/v) and 0.1% triethylamine as eluent. Fractions containing 4-HNE-DMA were identified by silica gel thin layer chromatography using the same mobile phase. Detection was realized by spraying a solution of 5% molybdatophosphoric acid in ethanol with 10% sulfuric acid and heating for 5 min in a Memmert laboratory-type drying cabinet (Schwabach, Germany) at 105 °C. Relevant fractions were combined, the solvent was evaporated in vacuo at 40 °C, and the product was lyophilized. 1H NMR (500 MHz, CDCl3) δ 0.86 (t, 3H, J = 7.9 Hz, H9), 1.23−1.33 (m, 6H, H-6, H-7, H-8), 1.50 (m, 2H, H-5), 3.30 (s, 6H, H-11, H-12), 3.45 (s, 1H, H-10), 4.13 (q, 1H, J = 6.3 Hz, H-4), 4.77 (d, 1H, J = 4.7 Hz, H-1), 5.63 (ddd, 1H, J = 1.3 Hz, J = 4.7 Hz, J = 15.8 Hz, H-2), 5.86 (ddd, 1H, J = 1.3 Hz, J = 6.0 Hz, J = 15.8 Hz, H-3). The compound was found to be pure by observance of a single spot on TLC. Yield = 346 mg (molar yield = 53.7%). Synthesis of (E)-5,5-Dideuterio-1,1-dimethoxynon-2-en-4-ol Dimethyl Acetal (4-HNE-DMA-d2). The precursors fumaraldehyde dimethyl acetal and deuterated pentylmagnesium bromide were synthesized according to a method published elsewhere.23 Therefore, 45.9 mL of acetone was given in a 100 mL three-neck flask, and 0.4605 g of amberlyst-15 catalyst and 0.339 mL of water were added. After the addition of 2.084 mL of fumaraldehyde bis(dimethyl acetal), the reaction was continued for 6 min at room temperature with stirring. After filtration through a bed of anhydrous sodium carbonate, the solvent was evaporated in vacuo at 40 °C to yield fumaraldehyde

between raw and roasted peanuts were detected by observing the UV absorbance. Additionally, the levels of Nε-carboxymethyllysine (CML) did not change either. This is in contrast to a study in which CML was directly quantified in raw and roasted peanuts by GC-MS,5 clearly showing that CML was not detectable in native peanuts, whereas CML levels increased depending on the roasting conditions. Up to now, direct and unambiguous quantification of individual, chemically characterized 4-HNE-derived lipation products has not been performed. The aim of this study, therefore, was to develop an HPLC-ESI-MS/MS method for the direct quantification of 4-HNE−lysine adducts in food proteins. For quantification of free 4-HNE in native and heated peanut oil and native and roasted peanuts, a GC-MS (EI) method was developed using synthesized deuterated 4-HNE as internal standard. To identify possible lipation products between 4-HNE and the ε-amino group of lysine, Nα-acetylL-lysine was incubated with 4-HNE and reaction products were identified by HPLC-ESI-MS/MS. After synthesis of 2pentylpyrrole lysine, this lipation product was directly quantified in peanuts by HPLC-ESI-MS/MS for the first time.

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MATERIALS AND METHODS

Materials. Pepsin (EC 3.4.23.1), Pronase E (EC 3.4.24.4), methanol, γ-nonalactone, ammonium chloride, sodium borohydride, and hydrochloric acid (37%) were obtained from Merck (Darmstadt, Germany). Leucine aminopeptidase (EC 3.4.11.1), prolidase (EC 3.4.13.9), dialysis tubing cellulose membrane (MWCO 14 kDa), tetradeuteromethanol, deuterochloroform, aluminum hydride, 1-octen3-ol, Hoveyda−Grubbs catalyst second generation, acrolein dimethylacetal, amberlyst 15 (hydrogen form), fumaraldehyde bis(dimethyl acetal), petroleum ether (boiling point 40−60 °C), diethyl ether, o(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride, bis(trimethylsilyl)trifluoroacetamide, triethylamine, pyridinium chlorochromate, and magnesium chips were obtained from Sigma-Aldrich (Taufkirchen, Germany). Nα-Acetyl-L-lysine and N-benzoylglycyl-Lphenylalanine were obtained from Bachem (Bubendorf, Switzerland). Dichloromethane, hexane, ethyl acetate (pure), and heptafluorobutyric acid were obtained from VWR (Darmstadt, Germany). Acetonitrile, chloroform, tetrahydrofuran, and sulfuric acid were obtained from Fisher Scientific (Schwerte, Germany). Monosodium phosphate, disodium phosphate, sodium sulfate, sodium carbonate, sodium hydroxide, ascorbic acid, sodium tetraborate, and magnesium sulfate were obtained from Grü s sing (Filsum, Germany). Tris(hydroxymethyl)aminomethane was obtained from Serva (Heidelberg, Germany). Thymol and acetone were obtained from Carl Roth (Karlsruhe, Germany). Pentane was from Th. Geyer (Renningen, Germany). Molybdatophosphoric acid was obtained from ChemPUR (Karlsruhe, Germany). 1,1-Dideuterio-1-bromopentane was from Campro Scientific (Berlin, Germany). All chemicals were of the highest purity, except otherwise indicated. For all experiments, ultrapure water was used, prepared by an ELGA LabWater Purelab Plus water system (Celle, Germany). For HPLC-ESI-MS/MS measurements, double-distilled water, prepared in the presence of potassium permanganate, was used. Refined peanut oil and roasted commercial peanuts were obtained from a local market. Raw peanuts were obtained from Veggie’s Delight (Düsseldorf, Germany). Preparation of Samples. Raw peanuts with shell were roasted in a ULM 500 laboratory oven (Memmert, Schwabach, Germany) for 20 and 40 min at 170 °C, respectively. Afterward, the samples were shelled, skinned, and crushed, using a kitchen machine. Commercially available, roasted peanuts were crushed analogously. Protein contents were analyzed by the Kjeldahl method using the factor 5.3 for oilseeds.19 Fifty grams of refined peanut oil was heated in 250 mL round-bottom flasks for 20 and 40 min at 170 °C, respectively. Nuclear Magnetic Resonance Spectroscopy (NMR). For nonane-1,4-diol (1,4-ND), 4-oxononanal (4-ON), and 2-pentylpyrrole 5274

DOI: 10.1021/acs.jafc.5b01502 J. Agric. Food Chem. 2015, 63, 5273−5281

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Journal of Agricultural and Food Chemistry dimethyl acetal. 1H NMR (400 MHz, CDCl3) δ 3.38 (s, 6H, H-5, H6), 5.07 (dd, 1H, J = 1.2 Hz, J = 3.9 Hz, H-4), 6.38 (ddd, 1H, J = 1.2 Hz, J = 7.8 Hz, J = 15.9 Hz, H-3), 6.64 (dd, 1H, J = 3.9 Hz, J = 15.9 Hz, H-2), 9.64 (d, 1H, J = 7.8 Hz, H-1). Purity = 93.1% (GC-MS). Yield = 1.2105 g (molar yield = 76.3%). Diethyl ether (0.8 mL) and 0.103 g of magnesium chips were given in a 25 mL three-neck flask. After the addition of 50 μL of 1,1-dideuterio-1-bromopentane, the reaction mixture was heated at 45 °C under reflux with stirring. The emerging white coloration indicated the starting reaction of the formation of the Grignard reagent deuterated pentylmagnesium bromide. Another 5.52 mL of diethyl ether were added followed by 350 μL of 1,1-dideuterio-1-bromopentane in 1.11 mL of diethyl ether, and the reaction mixture was heated under mild reflux at 45 °C for 30 min while the reaction mixture turned dark. After the reaction mixture had been cooled to 0 °C in an ice bath, 280 μL of fumaraldehyde dimethyl acetal in 1.84 mL of diethyl ether were added, and the reaction mixture was stirred for 30 min at 0 °C. By adding 2.76 mL of a solution of saturated ammonium chloride, the reaction was quenched and then extracted four times with 15 mL of diethyl ether, respectively. After drying over anhydrous sodium sulfate, the solvent was removed in vacuo at 40 °C. Purification of 4-HNE-DMA-d2 was realized as described for the undeuterated 4-HNE-DMA. 1H NMR (400 MHz, CDCl3) δ 0.87 (t, 3H, J = 6.4 Hz, H-9), 1.29 (m, 6 H, H-6, H-7, H-8), 3.31 (s, 6H, H-11, H-12), 3.46 (s, 1H, H-10), 4.13 (d, 1H, J = 6.3 Hz, H-4), 4.78 (d, 1H, J = 4.8 Hz, H-1), 5.64 (ddd, 1H, J = 0.9 Hz, J = 4.8 Hz, J = 15.7 Hz, H-2), 5.87 (ddd, 1H, J = 0.9 Hz, J = 6.0 Hz, J = 15.7 Hz, H-3). The compound was found to be pure by observance of a single spot on TLC. Yield = 154 mg (molar yield = 44.6%). GC-MS (EI) Quantification of Free 4-HNE in Peanut Oil and Peanut Samples. The developed method is partially comparable to a recently published method.4 The internal standard 4-HNE-d2 was obtained by acid hydrolysis of 2 mg of 4-HNE-DMA-d2 with 0.5 mL of 0.1 mol/L hydrochloric acid with stirring for 1 h in a 5 mL flask at room temperature. Following a 3-fold extraction with hexane (0.5 + 0.3 + 0.3 mL) to prohibit further release of free 4-HNE-d2 if the reaction was not complete, the concentration was calculated on the basis of the molar extinction coefficient for 4-HNE in hexane,1 ε = 14400 L mol−1 cm−1. To 1.0 g of crushed, shelled, and skinned peanuts or peanut oil were added 20 μL of an internal standard solution containing 95 nmol of 4-HNE-d2 and 10 mL of a solution of 0.1% ascorbic acid in water. The samples were homogenized using an ultraturrax at 11.200 rpm for 2 min, and the supernatant was collected after centrifugation at 10000g for 10 min at 4 °C. The extraction was repeated analogously, and the combined supernatants were extracted 3-fold with 10 mL of dichloromethane for 1 min, respectively. The organic phases were separated by centrifugation at 10000g for 2 min at 4 °C. After filtration (Whatman, 595 1/2) the dichloromethane was evaporated at 30 °C with nitrogen. The first derivatization step was realized by adding 200 μL of a o-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA-HCl) solution (20 mg/mL water) and heating in a water bath for 1 h at 40 °C. Hexane (1.3 mL) was added, and the samples were vortexed, followed by an addition of 100 μL of concentrated sulfuric acid24 and vortexing again. After centrifugation at 2100g for 3 min at room temperature, the hexane phase was removed, dried over anhydrous sodium sulfate, filtered (0.45 μm), and evaporated at 30 °C under nitrogen. The second derivatization step was performed at room temperature for 3 h by adding 200 μL of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). An aliquot was used for GC-MS (EI) analysis using an Agilent 7890A system, consisting of a 7683 series injector with a sample tray and a 5975C MS detector working in EI mode, all from Agilent (Böblingen, Germany), and a ZB-5 Guardian capillary column (30.0 m plus 5.0 m guard column, inner diameter = 0.25 mm, 0.25 μm film thickness) from Phenomenex (Torrance, CA, USA). Helium was used as carrier gas with a constant flow of 1.0 mL/min. With the injector temperature set to 250 °C, 1 μL of sample was injected by using the pulsed splitless mode. The auxiliary temperature was set to 250 °C, and the ion source and quadrupole were set to 230 and 150 °C, respectively. The initial oven temperature was set to 100 °C and held for 4 min and then raised at 15 °C/min to 300 °C. The post run time was set to 3 min at

300 °C. The mass spectrometer was working in electron impact mode at 70 eV. The solvent delay time was set to 10 min. SIM parameters were as follows: for 4-HNE (syn and anti), quantifier ion m/z 242 and qualifier ion m/z 352; and for 4-HNE-d2 (syn and anti), quantifier ion m/z 244 and qualifier ion m/z 352. The dwell time was set to 80 ms. Quantification was realized using authentic 4-HNE, being obtained from 4-HNE-DMA as previously described for 4-HNE-DMA-d2, the addition of 95 nmol of internal standard 4-HNE-d2, and an analogous derivatization procedure. Incubation of Nα-Acetyl-L-lysine with 4-HNE. For identification of major amino acid side chain reaction products formed between 4HNE and Nα-acetyl-L-lysine, 10 mM 4-HNE and 10 mM Nα-acetyl-Llysine were heated for 4 h at 75 °C in 5 mL of methanol in a 10 mL round-bottom flask under reflux. 4-HNE was obtained by acid hydrolyses as described above, and its concentration in aqueous solution was determined using the molar extinction coefficient1 ε = 13750 L mol−1 cm−1. One milliliter of the reaction mixture was taken for direct HPLC-ESI-MS/MS analysis. For reduction, 1.0 mL of the reaction mixture was evaporated under nitrogen, and 3.0 mL of a 0.2 M sodium tetraborate solution (pH 9.5) and 2.0 mL of a 1.0 M sodium borohydride solution in 0.1 M sodium hydroxide were added. After reduction for 17 h at room temperature, 1.0 mL of 6 M hydrochloric acid and 5 mL of 12 M hydrochloric acid were added, and the samples were hydrolyzed for 23 h at 110 °C. Afterward, the hydrochloric acid was removed by using a waterjet pump at 40 °C. The residue was dissolved in 1.0 mL of methanol and analyzed by HPLC-ESI-MS/MS. Incubation of Peanut Proteins with 4-HNE. Peanut proteins of raw peanuts were extracted as described elsewhere,6,25 except for using non-defatted peanuts instead of defatted peanuts. Approximately 50 mg of peanut protein extract was dissolved in 25 mL of 0.1 M phosphate buffer (pH 7.4), and 4-HNE, obtained from 4-HNE-DMA, was added in the following molar ratios related to the sum of relevant reactive amino acids cysteine, histidine, lysine, and arginine:6 0.1:1, 0.2:1, 1:1, and 5:1. Additionally, a blank sample consisting of peanut proteins without 4-HNE was prepared. The incubation was carried out under nitrogen for 24 h at 37 °C with stirring, followed by a dialysis (14 kDa MWCO) against deionized water for 24 h at 6 °C. Afterward, the samples were lyophilized and protein contents were analyzed by the Kjeldahl method using the factor 5.3 for oilseeds.19 For analysis of amino acid modification rates of cysteine, lysine, histidine, and arginine, the samples were hydrolyzed enzymatically. For analysis of 2PPL, the samples were hydrolyzed enzymatically, freeze-dried, and redissolved in 400 μL of water and 100 μL of methanol. The 0.1:1 sample was diluted 100-fold using water/methanol (80:20, v/v), whereas the other samples were diluted 500-fold including 40 μL of internal standard N-benzoylglycyl-L-phenylalanine in methanol/water (50:50, v/v) with a final concentration of 3.06 pmol/mL sample. The blank sample was used without dilution. After filtration (0.45 μmol), samples were subjected to HPLC-ESI-MS/MS analysis. Quantification was realized using authentic 2-PPL and internal standard Nbenzoylglycyl-L-phenylalanine with a final concentration of 3.06 pmol/mL in an enzymatic hydrolysate matrix calibration of the native peanut protein extract, prepared analogously to the samples. Synthesis of 2-Amino-6-(2-pentyl-1H-pyrrol-1-yl)hexanoic Acid (2-PPL). Lithium aluminum hydride (2.6 g) was added to 150 mL of tetrahydrofuran in a 500 mL three-neck flask, and the solution was cooled by an ice bath. γ-Nonalactone (12.5 mL) in 50 mL of tetrahydrofuran was added while cooling continued. After the threeneck flask was allowed to warm to room temperature, the reaction mixture was heated for 3 h under reflux with stirring. After cooling in an ice bath, ice-cold water was added until the formation of hydrogen stopped. After filtration over magnesium sulfate and drying over magnesium sulfate overnight, the tetrahydrofuran was evaporated at 40 °C. 1H NMR of nonane-1,4-diol (600 MHz, CDCl3) δ 0.82 (t, 3H, J = 7.2 Hz, H-9), 1.23 (m, 4 H, H-7, H-8), 1.37 (m, 4H, H-3, H-6), 1.57 (m, 4H, H-2, H-5), 3.52 (m, 2H, H-1), 3.57 (m, 1H, H-4). Purity = 98.2% (GC-MS). Yield = 8.4473 g (molar yield = 67.5%). In a 500 mL three-neck flask 17.0 g of pyridinium chlorochromate was suspended in 100 mL of dichloromethane.26 Nonane-1,4-diol (8.4473 g) in 50 5275

DOI: 10.1021/acs.jafc.5b01502 J. Agric. Food Chem. 2015, 63, 5273−5281

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Journal of Agricultural and Food Chemistry mL of dichloromethane was added with stirring. The reaction mixture was kept for 2 h at room temperature, resulting in a dark coloration of the reaction mixture. Afterward, 150 mL of diethyl ether were added, and the reaction mixture was filtered over magnesium sulfate. The addition of diethyl ether followed by a filtration step was repeated until no precipitation occurred. After evaporation of the diethyl ether at 40 °C, 4-oxononanal was purified by silica gel chromatography using ethyl acetate/hexane (15:85, v/v) as eluent.9 Fractions containing 4oxononanal were detected by GC-MS (EI) and combined, and the solvent was evaporated at 40 °C. 1H NMR of 4-oxononanal (600 MHz, CDCl3) δ 0.85 (m, 6H, H-9), 1.26 (m, 9H, H-7, H-8), 1.55 (m, 3H, H-6), 2.43 (t, 2H, J = 7.5 Hz, H-5), 2.71 (m, 4H, H-2, H-3), 9.77 (s, 1H, H-1). Purity = 49.3% (GC-MS). Yield = 2.3225 g (molar yield = 13.9%). One gram of 4-oxononanal and 2.0 g of Nα-acetyl-L-lysine were dissolved in 25 mL of acetonitrile/water (1:1, v/v) in a 50 mL flask under nitrogen and reacted in a Paal−Knorr synthesis at room temperature for 72 h. Afterward, the solvent was evaporated at 40 °C. Hydrolysis of the acetyl group was realized by adding 250 mL of a 2 M sodium hydroxide solution and heating for 23 h under reflux. The solvent was evaporated at 60 °C, and the residue was extracted three times with 10 mL of chloroform, respectively, filtered over magnesium sulfate, and dried over magnesium sulfate overnight. The solvent was evaporated at 40 °C, and 2-PPL was purified using a semipreparative HPLC system. The system consisted of a Smartline solvent manager 5000, a Smartline pump 1000, and a Smartline UV detector 2500 (all from Knauer, Berlin, Germany). Twenty-five milligrams of the unpurified reaction mixture were dissolved in 1.0 mL of methanol and separated after filtration (0.45 μm) using an Eurospher 100-10 C18 column (250 × 16 mm; Knauer, Berlin, Germany) at 23 °C (room temperature), a flow rate of 4.0 mL/min, and a detection wavelength of 227 nm. A gradient was used with solvent A (water) and B (acetonitrile). The gradient program started with 10% solvent B for 2 min, increased to 95% B within 28 min, isocratic elution with 95% B for 10 min, decrease to 10% B within 2 min, followed by an isocratic elution at 10% B for 10 min. 2-PPL eluted between 21 and 23 min. Multiple separations were performed, relevant fractions were combined, and finally the solvent was evaporated at 40 °C prior to HPLC-ESI-MS/MS and one- and two-dimensional NMR analysis. ESI-MS (positive mode), [M + H]+ m/z 267.3; 1H NMR (600 MHz, MeOD) δ 0.99 (m, 6H, H-15), 1.44 (m, 6H, H-14, H-13), 1.51 (m, 5H, H-7), 1.68 (m, 2H, H-12), 1.80 (m, 2H, H-6), 1.98 (m, 2H, H8a,b), 2.6 (t, 2H, J = 7.5 Hz, H-11), 3.56 (t, 1H, J = 6.0 Hz, H-9), 3.91 (t, 1H, J = 7.5 Hz, H-5), 5.8 (m, 1H, J = 3.0 Hz, H-2), 6.00 (t, 1H, J = 3.0 Hz, H-3), 6.64 (d, 1H, J = 3.0 Hz, H-4). 2-PPL eluted chromatographically pure by HPLC-ESI-MS analysis. Purity based on elemental analysis = 75.1%. Yield = 13.7 mg (molar yield = 4.4%). Quantitation of 2-PPL in Roasted Peanuts. For quantification of 2-PPL in peanuts, 35 mg of crushed raw, roasted or commercially available peanuts were hydrolyzed enzymatically using the 3-fold amounts of enzymes and solutions as described above. For evaluating amino acid release after enzymatic hydrolysis, the amounts of valine and leucine were compared to those obtained after acid hydrolysis before and after defatting of the samples. Afterward, the samples were freeze-dried and redissolved in 250 μL of water, 80 μL of methanol, and 20 μL of internal standard N-benzoylglycyl-L-phenylalanine in methanol/water (50:50, v/v) with a final concentration of 0.57 pmol/ mL. After filtration (0.45 μm), samples were subjected to HPLC-ESIMS/MS analysis. Quantification was realized using authentic 2-PPL and internal standard N-benzoylglycyl-L-phenylalanine with a final concentration of 0.57 pmol/mL in an enzymatic hydrolyzate matrix calibration of raw peanuts, prepared analogously to the samples. HPLC-ESI-MS/MS Analysis. The HPLC system consisted of a degasser, a pump, an autosampler, and a diode array detector, all from Agilent Technologies 1200 series (Böblingen, Germany). A triple quad LC-MS 6410 from Agilent Technologies was used for MS/MS measurements. For identification of major reaction products between 4-HNE and Nα-acetyl-L-lysine, 10 μL of samples were injected and separated using a Eurospher 100-5 C18 column (250 × 3.0 mm; Knauer) at 30 °C, a flow rate of 0.38 mL/min, and a detection wavelength of 227 nm. A gradient was used with solvent A (water) and

B (acetonitrile), each containing 2 mM heptafluorobutyric acid and 11 mM acetic acid. The gradient increased from 2 to 70% B within 50 min and to 90% B within 15 min, isocratic elution at 90% B for 10 min, decreased to 2% B within 3 min, and isocratic elution at 2% for 10 min. First, full scan analyses and then product ion scans were performed. Full scan analyses were performed from 5.1 to 78.0 min in positive mode, scan ranges were from m/z 80 to 1000, scan time was 500 ms, fragmentor voltage was 135 V, gas temperature was 300 °C, gas flow was 11 L/min, and nebulizer pressure was 15 psi. Product ion scans were performed for the unhydrolyzed samples from 15 to 45 min with precursor ions m/z 365.3 for the pyridinium derivative, m/z 327.3 for the Schiff base, m/z 309.2 for the 2-pentylpyrrol-acetyllysine, and m/z 287.2 for the reduced and cyclized Michael adduct after reduction and acid hydrolyses using the same conditions as described above. Product ion scan ranges were from m/z 60 to 400 with a scan time of 200 ms, a fragmentor voltage of 135 V, and collision voltages of 25, 20, 25, and 15 V for the pyridinium derivative, Schiff base, 2-pentylpyrrolacetyllysine, and the reduced and cyclized Michael adduct, respectively. For quantification of 2-PPL in modified peanut proteins and in peanut samples, 40 and 60 μL of samples, respectively, were separated using a Zorbax SB-C18 column (50 × 2.1 mm) from Agilent Technologies at 30 °C and a flow rate of 0.25 mL/min. A gradient was used with solvent A (water) and B (acetonitrile), each containing 2 mM heptafluorobutyric acid and 11 mM acetic acid. The gradient was as follows: 2% B was held for 5 min, increased to 51% within 17 min, increased to 90% B within 2 min, isocratic elution at 90% B for 6 min, decreased to 2% B within 1 min, and isocratic elution at 2% B for 9 min. Measurements were performed using the multiple reaction monitoring (MRM) mode in positive mode, gas temperature was set to 300 °C, gas flow was set to 11 L/min, and nebulizer pressure was set to 15 psi. Internal standard N-benzoylglycyl-L-phenylalanine was analyzed from 15.5 to 20.6 min with a mass transition from m/z 327.1 to 166.1 for quantification using fragmentor and collision voltages of 90 and 4 V, respectively, and a dwell time of 300 ms. Mass transition from m/z 327.1 to 105.1 was used for qualification using fragmentor and collision voltages of 90 and 34 V, respectively, and a dwell time of 300 ms. 2-PPL was analyzed from 20.6 to 28 min with a mass transition from m/z 267.1 to 204.2 for quantification using fragmentor and collision voltages of 148 and 18 V, respectively, and a dwell time of 100 ms. Mass transition from m/z 267.1 to 84.1 was used for qualification using fragmentor and collision voltages of 148 and 29 V, respectively, and a dwell time of 100 ms. Statistical Treatment. Results are expressed as mean values ± standard deviations of two separate measurements,27 except otherwise indicated.

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RESULTS AND DISCUSSION Quantitation of Free 4-HNE in Heated Peanut Oil and Peanut Samples. As reference material for the quantification of free 4-HNE in heated peanut oil and peanut samples, a deuterated 4-HNE-DMA-d2 standard was synthesized (see Supporting Information Figure I). Figure 1 shows the observed

Figure 1. Structure of synthesized 4-HNE-DMA-d2, showing the observed relevant heteronuclear multiple bond correlations (HMBCs).

relevant HMBCs of 4 HNE-DMA-d2. After development of a GC-MS (EI) method, the amounts of 4-HNE were quantified in peanut oil and peanut samples. Figure 2 (left) shows the amounts of free 4-HNE in a native and refined peanut oil as well as in a peanut oil after heating under roasting conditions (20 and 40 min at 170 °C). The amounts were between 0.1 and 5276

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Figure 2. Quantitation of free 4-HNE by GC-MS (EI): (left) amounts of 4-HNE in peanut oil heated at 170 °C; (right) amounts of 4-HNE in peanuts roasted at 170 °C.

0.6 μmol/100 g oil or between 0.2 and 1.0 μg/g oil, clearly indicating an increase in free 4-HNE depending on the heating time. These data are in agreement with published values for 4HNE in heated soybean oil, butter oil, and corn oil, resulting from HPLC analysis.28,29 For olive oil (extra virgin), amounts ranging from 0.06 to 0.12 μg 4-HNE/g oil were found using GC-MS.4 After methyl linoleate had been heated at 185 °C for up to 6 h, it was observed by HPLC analysis that within the first hour of heating, most of the 4-HNE had been formed.30 The amounts of the precursor linoleic acid in peanut oil and in peanuts are 21.6 and 14%, respectively,2 indicating that most of the 4-HNE may be formed in the initial stage of heating. This corresponds with the results in Figure 2 (left) and the more pronounced increase in 4-HNE formation during the first 20 min of roasting. Raw peanuts were roasted analogously (20 and 40 min at 170 °C), and the amounts of free 4-HNE are shown in Figure 2 (right). A decrease in free 4-HNE from 12.37 μmol/ 100 g protein (4.47 mg/kg peanut) in the native to 2.22 μmol/ 100 g protein (0.82 mg/kg peanut) in the roasted samples was found. The formation of 4-HNE in native peanuts could be a result of drying processes and storage of peanuts after harvesting. Commercially available, roasted peanuts contained 4-HNE in amounts of 2.88 μmol/100 g protein (1.29 mg/kg peanut). No difference between the samples roasted for 20 and 40 min was detectable, indicating that at this stage the formation and degradation of 4-HNE is in equilibrium. This fits with the amount of 4-HNE found in the commercial peanut sample, for which the starting amount is not known. Recently published results for 4-HNE in two commercially available peanut samples were between 0.12 and 0.60 mg/kg peanut sample, analyzed by GC-MS,4 which is comparable to the amounts of 4-HNE found in this study. Also, 0.14−0.41 mg 4HNE/kg of sample was found in walnuts and 0.21 mg/kg in an infant formula.4 In beef and pork, amounts ranging from 2.19 to 21.87 mg/kg and from 0.16 to 23.75 mg/kg, respectively, were found by HPLC analysis,31 whereupon a correlation between the amounts of 4-HNE and ω-6 fatty acids contents in pork was observed. In fresh blue mackerel, yellowtail, and chicken, amounts of 1.16, 0.50, and 0.04 mg/kg were found, respectively, analyzed by HPLC.32 A general statement about the toxicology of these levels of 4-HNE is not possible, because data about 4-HNE amounts reaching the small intestine after a gastrointestinal digestion are not available. Due to the fact that in the absence of further peanut constituents heated peanut oil shows an increase of 4-HNE, further reactions with nucleophilic amino acid side chain groups are supposed to be responsible for the loss of 4-HNE when whole peanuts are roasted. These

lipation reactions6 may contribute to the loss of lysine, which cannot be explained by the formation of Maillard reaction products.5 Amino Acid Modifications in Modified Peanut Proteins. To investigate the reactions of 4-HNE with nucleophilic amino acid side chains, extracted peanut proteins of raw peanuts were incubated with 4-HNE at 37 °C for 24 h in phosphate buffer (pH 7.4) as previously described for the incubation of other secondary products.6,33−35 Incubations were performed in different molar ratios between 4-HNE and the sum of the reactive amino acids cysteine, histidine, lysine, and arginine. Samples were hydrolyzed enzymatically because of the instability of certain possible reaction products toward acid hydrolysis. Figure 3 shows the decreases of the amino acids

Figure 3. Decreases of amino acids cysteine, histidine, lysine, and arginine in extracted peanut proteins modified by 4-HNE for 24 h at 37 °C in 0.1 M phosphate buffer, analyzed by amino acid analysis. Decreases are presented relative to the amounts of the blank sample.

cysteine, histidine, lysine, and arginine relative to the amounts in the blank sample. Lysine modifications increased from 7.5 to 25.3%, whereas histidine, cysteine, and arginine remained unchanged. Due to the fact that cysteine is able to form disulfide bonds, its accessibility for 4-HNE may be limited, although its reactivity toward 4-HNE is higher than that of lysine.9 Identification of Major Reaction Products of 4-HNE and Nα-Acetyl-L-lysine. For identification of major reaction products of 4-HNE and the ε-amino group of lysine, 4-HNE and Nα-acetyl-L-lysine were incubated in equimolar amounts in methanol for 4 h at 75 °C. Lipation products were identified by HPLC-ESI-MS/MS analysis before hydrolysis (Figure 4). The Michael adduct was identified due to its instability36 in its reduced, cyclized form after reduction and acid hydrolysis 5277

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Figure 4. Identification of lipation products by HPLC-ESI-MS/MS after incubation of Nα-acetyl-L-lysine and 4-HNE in equimolar amounts in methanol for 4 h at 75 °C. The HPLC-ESI-MS-scan chromatogram (top) shows peaks of Nα-acetyl-L-lysine (m/z 189.2), a pyridinium derivative (m/ z 365.2), a Schiff base (m/z 327.2), and a 2-pentylpyrrol adduct (m/z 309.2). (Middle) Product ion patterns after fragmentation of the derivatives and selected ions (bottom) resulting from the derivatives, respectively. Detailed explanations of the ions are given in the text.

is indicative for the pyrrol moiety. The characteristic fragment m/z 204.2 might result from a cyclization step analogous to the Schiff base. A possible formation pathway of the characteristic ion with m/z 204.2 is given in Figure III in the Supporting Information, based on the proposed fragmentation of adducts between the ε-amino group of lysine and hexanal.38 Formation of the 2-pentylpyrrol derivative occurs initially by the formation of a Schiff base, being in an equilibrium with its enamine, which undergoes keto−enol tautomerism to the 4oxo-enamine derivative that finally cyclizes to the 2pentylpyrrol derivative.9 After reduction and acid hydrolysis, a monoisotopic mass of m/z 287.2 [MH]+ and fragments m/z 242.2 (loss of carboxyl group), m/z 224.2 (losses of carboxyl and amino groups), m/z 158.2 (cleavage between δ carbon and ε nitrogen), and, as typical lysine fragments, m/z 84.1 and 130.1 (Figure II in the Supporting Information) were observed. This points to the presence of the reduced, cyclized Michael adduct. Due to the fact that 2-PPL is an advanced lipation end product,8,11 further studies were performed. For synthesis of 2PPL (for the reaction scheme see Figure IV in the Supporting Information), γ-nonalactone was reduced to nonane-1,4-diol by LiAlH4, which was oxidized to 4-oxononanal by pyridinium chlorochromate. Following a Paal−Knorr synthesis including Nα-acetyl-L-lysine, the acetyl group was removed by basic hydrolysis using a 2 M sodium hydroxide solution. Purification

(Figure II in the Supporting Information). The monoisotopic mass of m/z 365.2 [M]+ and characteristic fragments of the pyridinium moiety (m/z 194.2) after additional loss of water (m/z 176.1) are indicative for a 4-HNE-lysine pyridinium derivative. Furthermore, the fragmentation pattern shows typical lysine fragments (m/z 84.2 and 130.1). Ions with m/z 323.1 and 278.1 are indicative for secessions of the acetyl group and the carboxyl and acetyl groups, respectively. After incubation of 4-hydroxypentenal and glycine, an analogous pyridinium derivative was detected, and a possible reaction pathway was proposed37 requiring two molecules of 4hydroxypentenal and one molecule of glycine. To the best of our knowledge, this is the first report of a pyridinium derivative resulting from 4-HNE and the ε-amino group of lysine. Therefore, its formation is also possible in proteins. Further strong hints indicating the presence of a Schiff base were found: a monoisotopic mass of m/z 327.2 [MH]+ and characteristic fragments m/z 240.3 indicating losses of the acetyl and carboxyl groups, m/z 267.3, indicating a secession of the α-amino group with its acetyl moiety, and m/z 222.2, which results after cyclization as analogously postulated for free lysine.38 The fragment with m/z 204.2 is indicative for loss of water from the cyclized ion. With fragments m/z 267.2 and 222.2, the monoisotopic mass m/z 309.2 [MH]+ shows typical ions of the 2-pentylpyrrole-acetyl lysine (2-PPAcL) indicating losses of the acetyl and carboxyl groups, respectively. The ion m/z 138.2 5278

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HNE may occur, so that less 4-HNE would be available for the reaction with lysine. In further studies the amounts of free 4HNE could be measured to check whether the amounts of free 4-HNE are really lowered in the 1:1 and 5:1 samples. Supposing that 2-PPL would be the kinetically preferred reaction product, the reaction pathway could shift at higher concentrations so that, for example, the reaction can remain on the level of the Schiff base, the precursor of 2-PPL. Quantitation of 2-PPL in Peanuts. Due to its instability during acid hydrolysis, 2-PPL was released from the peanut protein samples enzymatically. To ensure complete enzymatic degradation, results for the amounts of valine and leucine obtained after enzymatic hydrolysis for the native, the two roasted, and the commercial samples were compared to the corresponding values after acid hydrolysis. For valine the enzymatic release ranged from 87.5 to 103.6% and for leucine from 83.4 to 97.0%, indicating complete enzymatic hydrolysis. Quantitation of 2-PPL was realized by HPLC-ESI-MS/MS (MRM mode). Figure 7 shows a representative HPLC-ESI-

was realized by semipreparative HPLC with UV detection, using an RP-18 column. Identification of 2-PPL was performed by HPLC-ESI-MS/MS measurements and one- and twodimensional NMR spectroscopy. Figure 5 shows the structure of 2-amino-6-(2-pentyl-1H-pyrrol-1-yl)hexanoic acid (2-PPL) and the relevant HMBCs.

Figure 5. Structure of 2-PPL showing the observed relevant heteronuclear multiple bond correlations (HBMC).

Quantitation of 2-PPL in Modified Peanut Proteins. 2PPL was quantified after enzymatic hydrolysis by HPLC-ESIMS/MS. Figure 6 shows the decreases of lysine compared to

Figure 7. Representative HPLC-ESI-MS/MS chromatogram (MRM mode, transition m/z 267.1 → 204.2) of the enzymatic hydrolysate of the commercial peanut sample (black) and the 2-PPL standard in a raw peanut hydrolyzate matrix (gray).

MS/MS chromatogram (m/z 267.1 → 204.2) of the hydrolyzed commercial peanut sample and the 2-PPL standard in a raw peanut hydrolyzate matrix. The quantitated amounts of 2PPL in raw, roasted, and commercial peanut samples are given in Figure 8. In raw peanuts, 2-PPL was not detectable, whereas in roasted peanuts, the amounts ranged from 112.1 to 175.1 nmol/100 g protein or from 29.9 to 46.7 μg/100 g protein. In the commercial sample, 144.5 nmol/100 g protein or 38.5 μg/ 100 g protein was found. These results point to an increase of 2-PPL depending on the roasting conditions. Data obtained for the commercial sample and the samples roasted in laboratory scale were comparable. It should be noted that the formation of 2-PPL would also be possible by a Paal−Knorr reaction if 4oxononanal was present. Compared to the loss of free 4-HNE as a result of peanut roasting, approximately 1 and 2% of 4HNE could be recovered, bound to the ε-amino group of lysine as 2-PPL in the peanut samples roasted for 20 and 40 min, respectively. In this estimation, the formation of 4-HNE during the roasting process could not be taken into account. Due to the fact that at low 4-HNE concentrations the total loss of lysine can be explained by the formation of the lipation product

Figure 6. Decrease of lysine due to modification of extracted peanut proteins by 4-HNE after incubation at 37 °C for 24 h in 0.1 M phosphate buffer, analyzed by amino acid analysis compared to the lysine level in the blank sample and amounts of 2-PPL quantitated after enzymatic hydrolysis by HPLC-ESI-MS/MS.

the blank sample analyzed by amino acid analysis and the formation of 2-PPL analyzed by HPLC-ESI-MS/MS, each after enzymatic hydrolysis. In the blank sample, 2-PPL was not detectable. The results indicate that 2-PPL is readily formed when 4-HNE is present and only a slight increase can be observed when the ratio of 4-HNE is increased. In the 5:1 sample, only 12% of the 2-PPL amount compared to the 0.1:1 sample was quantified. These results show that the amounts of 2-PPL as well as the loss of lysine do not increase depending on the amount of 4-HNE. Furthermore, it can be seen that at low concentrations of 4-HNE (0.1:1 and 0.2:1 samples), 2-PPL is the major reaction product, explaining the total loss of lysine, whereas at higher 4-HNE concentrations (1:1 and 5:1 samples), other reaction products must be formed. At higher concentrations of 4-HNE, a polymerization or oxidation of 45279

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ASSOCIATED CONTENT

S Supporting Information *

Figures I−IV. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.jafc.5b01502.

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AUTHOR INFORMATION

Corresponding Author

*(T.H.) Phone: +49-351-463-34647. Fax: +49-351-463-34138. E-mail: [email protected]. Notes

Parts of this study were presented as poster contributions to the 43rd Lebensmittelchemikertag, September 22−24, 2014, Gießen, Germany. The authors declare no competing financial interest.

Figure 8. Quantitated amounts of 2-PPL in raw and roasted (170 °C) peanut samples as well as in a commercial peanut sample by HPLCESI-MS/MS after enzymatic hydrolysis. n.d., not detectable, (n = 3).

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ACKNOWLEDGMENTS We thank Karla Schlosser, Institute of Food Chemistry, TU Dresden, for performing the amino acid analysis; Anke Peritz, Institute of Organic Chemistry, TU Dresden, for performing the elemental analysis; and Dr. Margit Gruner, Institute of Organic Chemistry, TU Dresden, and Sivathmeehan Yogendra, Institute of Inorganic Chemistry, TU Dresden, for performing the NMR analysis.

2-PPL (Figure 6), other 4-HNE−lysine adducts should not be of importance. With regard to the loss of amino acids as a result of a modification of raw peanut proteins by 4-HNE (Figure 3), reactions of 4-HNE with other amino acid side chains besides the ε-amino group of lysine seem not to be relevant. A slight decrease in histidine was observed, which might be explained by the formation of Michael adducts or its resulting hemiacetal.39 Besides the one-to-one 4-HNE−amino acid adducts, crosslinking products may also contribute to the loss of lysine, caused by fluorophores or a Schiff base and Michael adduct at the same molecule of 4-HNE.12,13 Heating of 4-HNE and primary amines results in only trace amounts of pyrrole and mainly in the condensation product of 4-HNE, 2-pentylfuran.9 This might, in contrast to peanut oil, contribute to a loss of 4HNE when proteins are present. In conclusion, our results show that 4-HNE, a well-known secondary product of the autoxidation of ω-6 polyunsaturated fatty acids,1 is formed as a result of heating peanut oil depending on the roasting time. Roasting whole peanuts, however, led to a significant decrease in 4-HNE, due to reactions with the amino acid side chains of the peanut proteins as well as a possible degradation of 4-HNE to 2-pentylfuran. As shown, especially lysine derivatives are formed, among which a novel identified pyridinium derivative, Schiff bases, Michael adducts, and 2-pentylpyrrol adducts represent potential lipation products. For a model system, consisting of extracted peanut proteins incubated with 4-HNE at 37 °C for 24 h, the complete decrease of lysine could be explained by the formation of 2-PPL at low concentrations of 4-HNE, indicating that 4-PPL is a good marker for a protein modification by 4-HNE. Furthermore, to the best of our knowledge, a direct quantification of 2-PPL was performed for the first time, showing an increase in 2-PPL depending on the roasting time. This post-translationally formed lipation product contributes to a small percentage of the total loss of lysine observed when peanuts are roasted,5 showing that lipation products are formed in the course of peanut roasting. Considering the broad spectrum of possible precursors formed when lipid-rich peanuts are roasted, a complex spectrum of lipation products can be formed, accounting for the large modification rate of lysine.6 Further studies are needed to quantify the amounts of other 4HNE−lysine adducts and to investigate the influence of these adducts on the allergenic potential of roasted peanuts.

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ABBREVIATIONS USED 4-HNE, 4-hydroxynon-2-enal; MWCO, molecular weight cutoff; GC-MS (EI), gas chromatography with mass spectrometry after electron-impact ionization; HPLC-ESI-MS/MS, highperformance liquid chromatography with electrospray ionization and tandem mass spectrometry; 2-PPL, 2-amino-6-(2pentyl-1H-pyrrol-1-yl)hexanoic acid (2-pentylpyrrole lysine)

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REFERENCES

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NOTE ADDED AFTER ASAP PUBLICATION Due to a production error, this paper was published on the Web on May 18, 2015, with none of the requested galley corrections implemented. The corrected version was reposted on May 19, 2015.

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DOI: 10.1021/acs.jafc.5b01502 J. Agric. Food Chem. 2015, 63, 5273−5281