Oxidative versus Non-oxidative Decarboxylation of ... - ACS Publications

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Journal of Agricultural and Food Chemistry

Oxidative vs. Non-oxidative Decarboxylation of Amino Acids: Conditions for the Preferential Formation of Either Strecker Aldehydes or Amines in Amino Acids/Lipid-derived Reactive Carbonyls Model Systems Rosario Zamora, M. Mercedes León and Francisco J. Hidalgo* Instituto de la Grasa, Consejo Superior de Investigaciones Científicas, Carretera de Utrera km 1, Campus Universitario – Edificio 46, 41013-Seville, Spain

Corresponding author: Francisco J. Hidalgo Instituto de la Grasa, CSIC Carretera de Utrera, km 1 Campus Universitario – Edificio 46 41013-Seville Spain

Phone: +34 954 611 550 Fax: +34 954 616 790 e-mail: [email protected]

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ABSTRACT: Comparative formation of both 2-phenylethylamine and

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phenylacetaldehyde as a consequence of phenylalanine degradation by carbonyl

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compounds was studied in an attempt to understand if the amine/aldehyde ratio can be

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changed as a function of reaction conditions. The assayed carbonyl compounds were

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selected because of the presence in the chain of both electron donating and electron

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withdrawing groups and included alkenals, alkadienals, epoxyalkenals, oxoalkenals, and

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hydroxyalkenals, as well as lipid hydroperoxides. The obtained results showed that 2-

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phenylethylamine/phenylacetaldehyde ratio depended on both the carbonyls and the

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reaction conditions. Thus, it can be increased by using electron donating groups in the

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chain of the carbonyl compound, small amounts of carbonyl compound, low oxygen

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content, increasing the pH, or increasing the temperature at pH 6. Opposed conditions

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(use of electron withdrawing groups in the chain of the carbonyl compound, large

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amounts of carbonyl compound, high oxygen contents, low pH values, and increasing

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temperatures at low pH values) would decrease 2-phenylethylamine/phenylacetaldehyde

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ratio and the formation of aldehydes over amines in amino acid degradations would be

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favored.

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KEYWORDS: Amino acid degradation; Biogenic amines; Lipid oxidation; Maillard

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reaction; Reactive carbonyls; Strecker aldehydes; Strecker-type degradation

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INTRODUCTION

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Nonenzymatic browning reactions have important consequences on the nutritional

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and sensory properties of foods: both positive, like the formation of important taste and

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aroma compounds or the pleasant browning produced in some cooked foods; and

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negative, such as the loss of essential amino acids or the formation of potentially toxic

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compounds.1–6 Many of these consequences are related to amino acid degradations.

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Among them, the Strecker degradation of amino acids is a source of important volatile

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constituents of food flavors, including Strecker aldehydes, pyrazines, pyridines,

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pyrroles, and oxazoles, among other compounds.7 On the other hand, production of

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amines by amino acid degradation in the presence of reactive carbonyl compounds –

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which was firstly described by Schieberle’s group in the Maillard reaction8 and then

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extended to lipid-derived reactive carbonyls–9,10 is a cause of concern both because of

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their potential toxicity and their involvement in the formation of vinylogous derivatives

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of amino acids such as acrylamide.11,12

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Strecker aldehydes and amines are produced simultaneously in food products by

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parallel pathways that compart key intermediates. A detailed discussion of the pathways

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involved in the amino acid degradation produced by lipid-derived reactive carbonyls has

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been described by Hidalgo and Zamora.13 Figure 1 schematizes the main intermediates

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and pathways involved, including also the described conversion of amines into Strecker

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aldehydes through the corresponding imines.14 As can be observed, the reaction

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produces in a first step the imine, which is then decarboxylated. This decarboxylation

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can be better understood from the zwitterionic form of the α-iminocarbonyl compound.

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The distribution of the electronic density in the azomethine ylide produced after the loss

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of carbon dioxide will determine the product formed. This has important consequences

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in foods because it will decide whether the reaction will mainly evolve towards either 3 ACS Paragon Plus Environment


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the formation of flavors or the formation of amines, which eventually can be later

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transformed into vinylogous derivatives of amino acids.

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In an attempt to determine if the Strecker aldehyde/amine ratio in food products can

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be changed as a function of reaction conditions, this study analyzes the formation of 2-

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phenylethylamine and phenylacetaldehyde in the reaction of phenylalanine with

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different lipid-derived reactive carbonyls as a function of pH, concentration of the

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carbonyl compound, water activity, amount of oxygen in the reaction atmosphere, time,

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and temperature. This study also includes the formation of benzaldehyde because this

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aldehyde is produced by phenylacetaldehyde degradation.15 Therefore, its determination

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will provide a better understanding of the formation and fate of phenylacetaldehyde. To

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the best of our knowledge this is the first study suggesting that the Strecker

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aldehyde/amine ratio can be changed as a function of the reactive carbonyls involved

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and the reaction conditions. Furthermore, the produced changes in Strecker

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aldehyde/amine ratio can be mostly understood on the basis of their formation pathway.

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MATERIALS AND METHODS

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Chemicals. Different hydroperoxides and lipid-derived reactive carbonyls from ω–3

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and ω–6 fatty acids were employed in these studies. 13-Hydroperoxyoctadeca-9,11-

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dienoic acid (LOOH), methyl 13-hydroperoxyoctadeca-9,11-dienoate (LOOMe), and

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methyl 13-hydroperoxyoctadeca-9,11,15-trienoate (LnOOMe) were prepared by

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oxidation of the corresponding fatty acids with lipoxygenase and later esterification

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with diazomethane following a previously described procedure.16,17 2-Octenal (OC) and

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2,4-alkadienals [2,4-heptadienal (HD) and 2,4-decadienal (DD)] were purchased from

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Aldrich (Milwaukee, WI). 4,5-Epoxy-2-alkenals [4,5-epoxy-2-heptenal (EH) and 4,5-

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epoxy-2-decenal (ED)] were prepared by epoxidation of 2,4-alkadienals (2,4-

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heptadienal and 2,4-decadienal, respectively) with 3-chloroperoxybenzoic acid.18,19 44 ACS Paragon Plus Environment

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Oxo-2-alkenals [4-oxo-2-hexenal (OH) and 4-oxo-2-nonenal (ON)] were synthesized

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from 2-alkylfurans (2-ethylfuran and 2-pentylfuran, respectively) with N-

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bromosuccinimide.20,21 4-Hydroxy-2-nonenal was prepared according to the procedure

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of Gardner et al.22All other chemicals were purchased from Aldrich, Sigma (St. Louis,

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MO), Fluka (Buchs, Switzerland), or Merck (Darmstadt, Germany) and were analytical

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grade.

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Phenylalanine/oxidized lipid reaction mixtures. Model reactions were carried out

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analogously to the procedure of Zamora and Hidalgo,23 which was modified. Briefly,

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mixtures of phenylalanine and the lipid derivative (10 µmol of each) were singly

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homogenized with 50-70 mesh sand (600 mg) (Aldrich), 30 µL of 0.3 M buffer, and 80

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µL of water. Samples were heated under controlled atmosphere in closed test tubes at

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the indicated times and temperatures, usually 1 h at 140 ºC. After cooling (5 min at

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room temperature and 15 min at –30 ºC), 20 µL of internal standard (24.09 mg of

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ethylpyridine in 50 mL of methanol) and 1 mL of methanol-water (80:20) were added.

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The mixture was stirred for 1 min and centrifuged for 10 min at 2000 × g. Seven

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hundred microliters of the obtained supernatant were transferred to a new test tube and

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reduced with 1 mg of sodium borohydride for 30 min. After this time, 500 µL of

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acetone were added and the test tube was stirred and centrifuged for 10 min at 2000 × g.

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The produced compounds were determined by GC–MS. The ions monitored for the

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quantitation of the different analytes were: [C7H8N]+ = 106 for the internal standard,

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[C7H7]+ = 91 for the 2-phenylethylamine, [C8H10O]+ = 122 for the phenylacetaldehyde

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(determined as 2-phenylethanol), and [C7H8O]+ = 108 for the benzaldehyde (determined

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as benzyl alcohol).

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GC-MS analyses. GC-MS analyses were conducted with a Hewlett-Packard 6890

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GC Plus coupled with an Agilent 5973 MSD (mass selective detector, quadrupole type). 5 ACS Paragon Plus Environment


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A fused-silica CAM capillary column (30 m × 0.25 i.d.; coating thickness, 0.25 µm)

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was used, and 1 µL of sample was injected in the pulsed splitless mode. Working

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conditions were as follows: carrier gas, helium (1 mL/min at constant flow); injector,

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250 ºC; oven temperature programmed from 80 ºC (4 min) to 120 ºC at 2 ºC/min and

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then to 220 ºC at 15 ºC/min; transfer line to MSD, 280 ºC; ionization EI, 70 eV; ion

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source temperature, 230 ºC; mass range, 28–550 amu.

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Determination of 2-phenylethylamine, phenylacetaldehyde, and benzaldehyde

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contents. Quantitation of 2-phenylethylamine, phenylacetaldehyde (as 2-

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phenylethanol), and benzaldehyde (as benzyl alcohol) was carried out by preparing

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standard curves of these compounds in the 600 mg of sand containing 80 µL of water

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and 30 µL of sodium phosphate buffer, pH 6, and following the whole procedure

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described above (without heating). Ten different concentration levels of the determined

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compounds were used (0, 0.25, 0.5, 1, 2, 3, 4, 5, 7.5, and 10 µmol). 2-Phenylethylamine,

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phenylacetaldehyde, and benzaldehyde contents were directly proportional to the

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analyte/internal standard area ratio (r = 0.998, p< 0.0001). The coefficients of variation

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were less than 8%.

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Statistical analysis. All data given are mean ± SD values of, at least, three

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independent experiments. Statistical comparisons among different groups were made

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using analysis of variance. When significant F values were obtained, group differences

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were evaluated by the Tukey test.24 Statistical comparisons were carried out using

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Origin v. 7.0 (OriginLab Corporation, Northampton, MA). The significance level is p

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< 0.05 unless otherwise indicated.

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RESULTS AND DISCUSSION


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Formation of 2-phenylethylamine, phenylacetaldehyde, and benzaldehyde in

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phenylalanine/lipid oxidation product reaction mixtures.When mixtures of

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phenylalanine and lipid oxidation products were heated together, the formation of 2-

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phenylethylamine, phenylacetaldehyde and benzaldehyde was observed. The amount of

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the formed compounds depended on the reaction conditions and the lipid oxidation

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product involved. Table 1 shows the formation of these three compounds in the

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presence of different lipid oxidation products at two pH values (3 and 6) and in the

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presence of either nitrogen or air.

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As can be observed, phenylalanine was converted into 2-phenylethylamine with a

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reaction yield of 0–3%, which depended on the lipid involved, the pH of the reaction

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and the presence, or not, of oxygen. The highest yields were observed when the reaction

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was carried out in the presence of alkadienals. These compounds produced about 3% of

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the amine when the reaction was carried out under nitrogen and this yield was reduced

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to 1% or less when the reaction was carried out under air. Other good producers of 2-

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phenylethylamine were the assayed hydroperoxides. These compounds produced the

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amine with a yield of 1.4–2.6% at pH 3 and this yield was independent of the presence

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or not of oxygen. The yield decreased to about 1% at pH 6 in the presence of nitrogen

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and to about 0.2–0.8% in the presence of air at this pH. Other assayed lipids were worse

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producers of the amine and the obtained yields were usually lower than 1% with the

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exception of 2-octenal (1.5% at pH 3 under air and 2.4% at pH 6 under nitrogen) and

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4,5-epoxy-2-decenal (1.2% at pH 3 under air and 1.3% at pH 6 under nitrogen).

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Differently to 2-phenylethylamine, phenylacetaldehyde was produced to a higher

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extent under air than under nitrogen. At pH 3 under air many lipids produced more than

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10% of phenylacetaldehyde, including hydroperoxides (11–16%), alkadienals (8–13%),

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and epoxyalkenals (10–12%), but not oxoalkenals (4–7%) or 4-hydroxynonenal (1%). 7 ACS Paragon Plus Environment


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These yields decreased to less than 4% when the reaction was carried out under

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nitrogen. Phenylacetaldehyde was produced with a reaction yield of 2–5% at pH 6 under

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air and this yield decreased to < 2.5% when the reaction was carried out under nitrogen

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at this pH.

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A behavior similar to that of phenylacetaldehyde was also observed for

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benzaldehyde, which is in agreement with the production of benzaldehyde from

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phenylacetaldehyde as its main formation pathway.15 Nevertheless, benzaldehyde was

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produced to a lower extent than phenylacetaldehyde and there were not too much

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differences among the different lipid oxidation products. Thus, benzaldehyde was

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produced with a yield of 1–7% at pH 3 under air, and this yield decreased to 0.5–0.8%

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when the reaction was carried out under nitrogen at this pH. Analogously, benzaldehyde

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was produced with a yield of 1–4% at pH 6 under air, and this yield decreased to 0–

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0.3% when the reaction was carried out under nitrogen at this pH.

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All these changes, and mainly the changes in the produced 2-

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phenylethylamine/phenylacetaldehyde ratios, can be understood on the basis of the

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reaction pathway schematized in Figure 1. The obtained results showed that alkenals,

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alkadienals, hydroxyalkenals, and the linoleic acid hydroperoxide, but not the

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hydroperoxide methyl esters, were the compounds that produced the highest 2-

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phenylethylamine/phenylacetaldehyde ratios at both pH 3 and pH 6 under nitrogen.

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However, there was not a clear difference among the different lipid oxidation products

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when the reaction was carried out under air. This behavior is likely related to the role of

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the chain in the charge distribution of the azomethine ylide shown in Figure 1. Thus,

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under nitrogen, the presence of electron withdrawing groups in the chain, such in

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oxoalkenals or epoxyalkenals, favored a charge distribution closer to mesomer b and,

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therefore, the formation of the aldehyde. On the contrary, the presence of electron 8 ACS Paragon Plus Environment

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donating groups in the chain such as alkoxyl or carbon-carbon double bonds, favored a

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charge distribution closer to mesomer a, and, therefore, the formation of the amine. In

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the presence of oxygen, double bonds are expected to be oxidized and converted into

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electron withdrawing groups. Therefore, under air, there was not a so clear difference

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among the different assayed carbonyl compounds.

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In order to carry out a detailed study of the effect of reaction conditions on the

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formation of these amino acid degradation products, 2,4-decadienal was selected as the

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lipid oxidation product because it is a good producer of both 2-phenylethylamine and

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phenylacetaldehyde and the mechanism by which these two compounds are produced

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has been previously studied.9,25

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Effect of the percentage of oxygen in the atmosphere on the formation of 2-

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phenylethylamine, phenylacetaldehyde, and benzaldehyde in phenylalanine/2,4-

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decadienal reaction mixtures. As expected according to the data shown in Table 1, 2-

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phenylethylamine on one hand, and phenylacetaldehyde and benzaldehyde on the other,

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exhibited oppositing effects in relation to the presence of oxygen at both pH 3 and 6

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(Figures S1A and S1B, respectively, of the Supporting Information). 2-

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Phenylethylamine formation was very sensitive to the presence of oxygen, and, at pH 6,

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> 20% of oxygen completely inhibited its formation. At pH 3, although oxygen

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inhibited its formation, small amounts of 2-phenylethylamine were also produced in a

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100% atmosphere of oxygen. On the contrary, the amount of both phenylacetaldehyde

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and benzaldehyde usually increased as a function of the oxygen content in the

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atmosphere. Thus, at pH 3, the amount of both phenylacetaldehyde and benzaldehyde

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increased rapidly from 0–20% oxygen and then the amount phenylacetaldehyde

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remained more or less stable but the amount of benzaldehyde continued increasing but

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to a lower extent. Something similar occurred at pH 6. The concentration of both 9 ACS Paragon Plus Environment


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phenylacetaldehyde and benzaldehyde increased rapidly from 0–20% oxygen and then

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increased to a lower extent at a higher oxygen content. Therefore, the presence of

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oxygen rapidly shifted the 2-phenylethylamine/phenylacetaldehyde ratio towards the

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formation of the aldehyde (Figure 2A).

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As discussed above, in the presence of oxygen, double bonds should be oxidized and

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converted into electron withdrawing groups, consequently favoring a charge distribution

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closer to mesomer b and, then, the formation of the aldehyde. This effect was so

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important that the amount of phenylacetaldehyde increased by 4–5 times in relation to

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the amount of this compound produced in the absence of air (Table 1). This effect was

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also observed in the formation of benzaldehyde, although this compound was more

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dependent on oxygen and it increased about 5–6 times in relation to the amount of this

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compound produced in the absence of air at pH 3, and about 9–10 times in relation to

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the amount of this compound produced in the absence of air at pH 6. This higher

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dependence of benzaldehyde in the presence of air suggests an oxidative formation

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pathway for this last compound.

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Effect of reaction pH on the formation of 2-phenylethylamine,

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phenylacetaldehyde, and benzaldehyde in phenylalanine/2,4-decadienal reaction

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mixtures. 2-Phenylethylamine formation did not exhibit big changes as a function of

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reaction pH (Figure S2A of the Supporting Information), although it seemed to increase

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linearly (r = 0.85, p = 0.0017) as a function of pH from 2.3% at pH 2.15 to 2.8% at pH

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9. On the contrary, phenylacetaldehyde concentration decreased linearly (r –0.998, p
0.98, p< 0.0004) as a function of the amount of 2,4-decadienal added between 0 and

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10 µmol. Something similar occurred at pH 6 (Figure S3B of the Supporting

234

Information). 2-Phenylethylamine concentration increased rapidly from 0 to 4 µmol of

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2,4-decadienal and later remained unchanged. On the contrary, phenylacetaldehyde and

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benzaldehyde concentrations increased linearly (r > 0.94, p< 0.006) as a function of the

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amount of 2,4-decadienal added between 0 and 10 µmol. An explanation for this

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different behavior of the amine and the aldehydes as a function of the concentration of

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the carbonyl compound can be explained on the basis of the pathway shown in Figure 1.

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The formation of the amine is accompanied by the recovery of the initial reactive

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carbonyl. Therefore, small amounts of the reactive carbonyl will produce the amine and

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the reactive carbonyl will be recovered for producing more amine (the process is

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catalytic in nature). On the other hand, formation of the Strecker aldehyde and of 11 ACS Paragon Plus Environment


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benzaldehyde is accompanied by the destruction of the reactive carbonyl. Therefore,

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higher amounts of reactive carbonyls will increase the formation of the Strecker

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aldehyde.

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Considering the 2-phenylethylamine/phenylacetaldehyde ratio (Figure 2C), an

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increase in the amount of the carbonyl compound reduced linearly (r < –0.94, p < 0.015)

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this ratio at the two pH values studied. This is likely a consequence of both, the catalytic

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nature of the formation of the amine from reactive carbonyls described above, and the

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conversion of the amine into the aldehyde shown in Figure 1, a conversion favored at

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higher concentrations of the lipid-derived carbonyl.14

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Effect of amount of water added on the formation of 2-phenylethylamine,

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phenylacetaldehyde, and benzaldehyde in phenylalanine/2,4-decadienal reaction

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mixtures. The effect of the water added was different at pH 3 and 6. At pH 3 (Figure

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S4A of the Supporting Information), both 2-phenylethylamine and phenylacetaldehyde

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were produced to a higher extent when 70–80 µL of water were added. An increase in

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the amount of water decreased the amount of both compounds, and 2-phenylethylamine

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formation seemed to be more sensitive to the presence of large amounts of water than

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phenylacetaldehyde. Benzaldehyde formation also decreased when amount of water

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increased and there was not a maximum like the one observed for 2-phenylethylamine

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or phenylacetaldehyde. At pH 6 (Figure S4B of the Supporting Information), 2-

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phenylethylamine was produced to a higher extent with 0–50 µL of water and decreased

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afterwards. On the contrary, the amount of phenylacetaldehyde produced increased with

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the addition of water from 0 to 120 µL and remained stable afterwards. Finally, the

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amount of benzaldehyde seemed to increase slightly when the amount of water

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increased.


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Therefore, and different to the other analyzed factors, the amount of water added did

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not always have the same consequences on the 2-phenylethylamine/phenylacetaldehyde

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ratio (Figure 2D). Thus, this ratio remained more or less unchanged at pH 3 but it

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decreased exponentially at pH 6, a behavior that cannot seem to be easily understood

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only on the basis of the reaction pathway shown in Figure 1.

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Effect of reaction time and temperature on the formation of 2-phenylethylamine

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and phenylacetaldehyde in phenylalanine/2,4-decadienal reaction mixtures. The

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amount of 2-phenylethylamine formed increased linearly (r > 0.98, p < 0.01) as a

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function of time at the different assayed temperatures when phenylalanine was heated in

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the presence of 2,4-decadienal at both pH 3 (Figure S5A of the Supporting Information)

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and 6 (Figure S6A of the Supporting Information). Reaction rates at the different

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assayed temperatures were calculated using the equation

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[2-phenylethylamine] = [2-phenylethylamine]0 + kt

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where [2-phenylethylamine]0 represents the intercept, k is the rate constant, and t is the

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time. These rate constants were used in an Arrhenius plot for calculation of activation

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energy (Ea) of 2-phenylethylamine formation from phenylalanine in the presence of 2,4-

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decadienal at pH 3 (Figure 3A) and 6 (Figure 3B). The values obtained for Ea were 51

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kJ/mol at pH 3 and 65 kJ/mol at pH 6.

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Analogously, the amount of phenylacetaldehyde formed also increased linearly (r >

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0.98, p < 0.01) as a function of the time at the different assayed temperatures when

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phenylalanine was heated in the presence of 2,4-decadienal at both pH 3 (Figure S5B of

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the Supporting Information) and 6 (Figure S6B of the Supporting Information).

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Reaction rates at the different assayed temperatures were also calculated using an

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equation similar to the above described for 2-phenylethylamine. The obtained rate 13 ACS Paragon Plus Environment


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constants were used in an Arrhenius plot for calculation of Ea of phenylacetaldehyde

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formation from phenylalanine in the presence of 2,4-decadienal at pH 3 (Figure 3A) and

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6 (Figure 3B). The values obtained for Ea were 74 kJ/mol at pH 3 and 40 kJ/mol at pH

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6.

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Differently to 2-phenylethylamine and phenylacetaldehyde, benzaldehyde was

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produced to very low extent under the assayed conditions and it was not possible to

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determine its formation Ea.

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2-Phenylethylamine/phenylacetaldehyde ratios were more or less constant at each

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temperature but decreased with temperature at pH 3 (Figure 4A) and increased with

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temperature at pH 6 (Figure 4B), which might be related to the easiness of conversion

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of the amine into the aldehyde at the different pHs and temperatures.

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All these data, as well as all other data obtained in this study suggests that 2-

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phenylethylamine/phenylacetaldehyde ratio can be modified as a function of reaction

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conditions. Thus, it can be increased by using electron donating groups in the chain of

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the carbonyl compound, small amounts of carbonyl compound, low oxygen content,

307

increasing the pH, or increasing temperature at pH 6. Contrary conditions (use of

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electron withdrawing groups in the chain of the carbonyl compound, large amounts of

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carbonyl compound, high oxygen contents, low pH values, and increasing temperatures

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at low pH values) would decrease 2-phenylethylamine/phenylacetaldehyde ratio and the

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formation of aldehydes over amines in amino acid degradations would be favored.

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AUTHOR INFORMATION

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Corresponding author

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*Telephone: +34 954 611 550. Fax: +34 954 616 790. E-mail: [email protected].


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Funding

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This study was supported in part by the European Union (FEDER funds) and the Plan

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Nacional de I + D of the Ministerio de Economía y Competitividad of Spain (project

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AGL2012-35627).

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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We are indebted to José L. Navarro for technical assistance.

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ASSOCIATED CONTENT

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Supporting Information

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Figures S1–S6. This material is free of charge via the Internet at http://pubs.acs.org



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(9) Zamora, R.; Delgado, R. M.; Hidalgo, F. J. Formation of β-phenylethylamine as a consequence of lipid oxidation. Food Res. Int. 2012, 46, 321–325. (10) Hidalgo, F. J.; Navarro, J. L.; Delgado, R. M.; Zamora, R. Histamine formation by lipid oxidation products. Food Res. Int. 2013, 52, 206–213. (11) Schieberle, P.; Köhler, P.; Granvogl, M. New aspects on the formation and analysis of acrylamide. In Advances in Experimental Medicine and Biology, 561; Friedman, M., Mottram, D., Eds.; Springer-Verlag: Berlin, Germany, 2005; pp. 205–222. (12) Zamora, R.; Delgado, R. M.; Hidalgo, F. J. Conversion of 3-aminopropionamide and 3-alkylaminopropionamide into acrylamide in model systems. Mol. Nutr. Food Res. 2009, 53, 1512–1520. (13) Hidalgo, F. J.; Zamora, R. Amino acid degradations produced by lipid oxidation products. Crit. Rev. Food Sci. Nutr., in press. DOI: 10.1080/10408398.2012.761173. (14) Zamora, R.; Delgado, R. M.; Hidalgo, F. J. Chemical conversion of phenylethylamine into phenylacetaldehyde by carbonyl–amine reactions in model systems. J. Agric. Food Chem. 2012, 60, 5491–5496. (15) Chu, F. L.; Yaylayan, V. A. Model studies on the oxygen-induced formation of benzaldehyde from phenylacetaldehyde using pyrolysis GC-MS and FTIR. J. Agric. Food Chem. 2008, 56, 10697–10704. (16) Hidalgo, F. J.; Zamora, R.; Vioque, E. Syntheses and reactions of methyl (Z)-9,10epoxy-13-oxo-(E)-11-octadecenoate and methyl (E)-9,10-epoxy-13-oxo-(E)-11octadecenoate. Chem. Phys. Lipids 1992, 60, 225–233. (17) Zamora, R.; Gallardo, E.; Hidalgo, F. J. Model studies on the degradation of phenylalanine initiated by lipid hydroperoxides and their secondary and tertiary oxidation products. J. Agric. Food Chem. 2008, 56, 7970–7975.



(18) Hidalgo, F. J.; Zamora, R. Modification of bovine serum albumin structure following reaction with 4,5(E)-epoxy-2(E)-heptenal. Chem. Res. Toxicol. 2000, 13, 501–508. (19) Zamora, R.; Navarro, J. L.; Gallardo, E.; Hidalgo, F. J. Chemical conversion of αamino acids into α-keto acids by 4,5-epoxy-2-decenal. J. Agric. Food Chem. 2006, 54, 2398–2404. (20) Shimozu, Y.; Shibata, T.; Ojika, M.; Uchida, K. Identification of advanced reaction products originating from the initial 4-oxo-2-nonenal-cysteine Michael adducts. Chem. Res. Toxicol. 2009, 22, 957–964. (21) Zamora, R.; Alcon, E.; Hidalgo, F. J. Effect of lipid oxidation products on the formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in model systems. Food Chem. 2012, 135, 2569–2574. (22) Gardner, H. W.; Bartelt, R. J.; Weisleder, D. A facile synthesis of 4-hydroxy-2(E)nonenal. Lipids 1992, 27, 686–689. (23) Zamora, R.; Hidalgo, F. J. Contribution of lipid oxidation products to acrylamide formation in model systems. J. Agric. Food Chem. 2008, 56, 6075–6080. (24) Snedecor, G. W.; Cochran, W. G. Statistical Methods, 7th ed.; Iowa State University Press: Ames, IA, USA, 1980. (25) Zamora, R.; Gallardo, E.; Hidalgo, F. J. Strecker degradation of phenylalanine initiated by 2,4-decadienal or methyl 13-oxooctadeca-9,11-dienoate in model systems. J. Agric. Food Chem. 2007, 55, 1308–1314.


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FIGURE LEGENDS Figure 1. Reaction pathway for the formation of 2-phenylethylamine and phenylacetaldehyde by phenylalanine degradation in the presence of lipid-derived reactive carbonyls. Benzaldehyde is also produced, mostly by phenylacetaldehyde degradation under oxidative conditions. Figure 2. Effect of: (A) oxygen content in the reaction atmosphere; (B) pH; (C) aldehyde concentration; and (D) water on the 2-phenylethylamine/phenylacetaldehyde (PEA/PAC) ratio produced by phenylalanine degradation in the presence of 2,4decadienal in sodium citrate buffer, pH 3 () and sodium phosphate buffer, pH 6 (). Samples were heated for 1 h at 140 ºC. Figure 3. Arrhenius plot for 2-phenylethylamine () and phenylacetaldehyde () formation by phenylalanine (Phe) degradation in the presence of 2,4-decadienal. Equimolecular mixtures of both compounds (10 µmol of each) were heated under nitrogen for 1 h in: (A) sodium citrate buffer, pH 3; and (B) sodium phosphate buffer, pH 6. Figure 4. Effect of time and temperature on the 2phenylethylamine/phenylacetaldehyde (PEA/PAC) ratio produced by phenylalanine degradation in the presence of 2,4-decadienal in: (A) sodium citrate buffer, pH 3; and (B) sodium phosphate buffer, pH 6. Equimolecular mixtures of both compounds (10 µmol of each) were heated under nitrogen at the indicated times and temperatures. The temperatures assayed were: 100 (), 120 (), 140 (), 160 ºC (), and 170 ºC ().



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Table 1. 2-Phenylethylamine, phenylacetaldehyde and benzaldehyde produced in mixtures of phenylalanine with lipid oxidation productsa

lipid Phe LOOH LOOMe LnOOMe OC HD DD EH ED OH ON HN

2-phenylethylamine pH 3 nitrogen air 0.00 ± 4.83 ± 0.00 f 1.22 e 25.95 ± 25.89 ± 2.18 a,b 3.00 a 20.99 ± 13.77 ± 2.88 b,c 2.90 b,c 16.05 ± 14.67 ± 3.62 c,d 3.34 b,c 8.87 ± 15.00 ± 0.83 d,e 2.80 b 25.30 ± 7.95 ± 5.77 a,b 0.55 d,e 30.99 ± 11.23 ± 3.37 a 2.03b,c,d,e 4.24 ± 8.50 ± 1.42 e,f 0.69b,c,d,e 6.56 ± 12.36 ± 1.78 e,f 1.31 b,c,d 0.20 ± 8.18 ± 0.10 f 1.05 c,d,e 0.10 ± 7.07 ± 0.04 f 1.70 d,e 5.46 ± 5.48 ± 1.39 e,f 1.48 e

pH 6 nitrogen 5.71 ± 1.01 b,c 11.56 ± 1.12 b 10.60 ± 0.87 b 11.25 ± 2.07 b 23.98 ± 4.53 a 27.63 ± 4.59 a 27.22 ± 5.02 a 8.12 ± 0.84 b,c 13.63 ± 1.69 b 1.76 ± 0.56 c 0.83 ± 0.23 c 8.58 ± 0.61 b,c

air 0.83 ± 0.31 d 7.55 ± 1.22 a 3.58 ± 1.18 b 2.15 ± 0.40 b,c,d 2.74 ± 0.22 b,c 3.18 ± 0.71 b,c 3.95 ± 0.56 b 3.78 ± 0.70 b 3.57 ± 0.65 b 3.35 ± 0.33 b 1.18 ± 0.20 c,d 0.64 ± 0.12 d

phenylacetaldehyde pH 3 nitrogen air 0.45 ± 3.87 ± 0.19 e 0.46 g 20.75 ± 104.52 ± 5.50 c,d 6.85 b,c 33.30 ± 160.54 ± 3.46 a,b 32.76 a 42.17 ± 118.08 ± 9.83 a 7.43 b,c 3.34 ± 30.96 ± 0.26 e 5.03 f,g 18.80 ± 81.96 ± 4.01 c,d 9.88 c,d,e 21.07 ± 126.82 ± 6.37 c,d 13.58 a,b 28.10 ± 102.81 ± 5.33 b,c 9.91 b,c,d 28.12 ± 122.60 ± 1.19 a,b,c 5.20 b 29.18 ± 43.46 ± 1.81 a,b,c 3.48 e,f 11.25 ± 66.02 ± 2.12 d,e 2.09 d,e 0.00 ± 11.46 ± 0.00 e 2.04 f,g

pH 6 nitrogen 0.00 ± 0.00 e 5.55 ± 0.83 c,d 14.26 ± 0.80 b 14.72 ± 3.36 b 2.69 ± 0.16 d,e 2.49 ± 0.74 d,e 3.93 ± 0.95 d,e 6.37 ± 2.03 c,d 24.21 ± 2.20 a 12.74 ± 3.02 b 7.72 ± 1.15 c 0.24 ± 0.06 e

a

air 21.08 ± 3.72 d,e,f 31.71 ± 7.35 d,e 52.79 ± 6.60 a 49.04 ± 5.73 a,b,c 15.52 ± 2.58 e,f 14.07 ± 2.60 f 36.88 ± 6.00 b,c,d 34.29 ± 4.37 c,d,e 27.18 ± 6.11 d,e 52.27 ± 12.16 a,b 33.59 ± 3.82 d,e 22.45 ± 0.58 d,e,f

benzaldehyde pH 3 nitrogen air 2.27 ± 6.02 ± 0.18 a 0.92 f 6.37 ± 60.57 ± 1.32 a,b 4.71 a,b 6.33 ± 69.87 ± 1.43 a,b 8.70 a 5.49 ± 51.40 ± 0.59 a,b,c 4.52 b,c 5.52 ± 20.54 ± 0.43 a,b,c 5.62 d,e 6.88 ± 19.48 ± 1.61 a,b 1.11 d,e,f 7.79 ± 47.55 ± 1.64 a 6.60 c 5.52 ± 24.53 ± 1.10 a,b,c 4.03 d,e 5.56 ± 30.27 ± 0.74 a,b,c 0.43 d 6.92 ± 13.41 ± 0.83 a,b 1.52 e,f 5.51 ± 16.46 ± 1.34 a,b,c 1.52 d,e,f 4.56 ± 14.56 ± 0.50 b,c 3.51 e,f

pH 6 nitrogen 0.00 ± 0.00 e 1.69 ± 0.51a,b,c,d 3.14 ± 0.41 a 2.94 ± 0.78 a,d 0.54 ± 0.21 b,e 1.60 ± 0.43 b,c,d 2.23 ± 0.38 a,c,d 1.14 ± 0.24 b,c,e 2.67 ± 0.75 a,d 2.87 ± 0.85 a 1.93 ± 0.62 a,c,d 0.00 ± 0.00 e

air 3.43 ± 1.33 f 18.99 ± 3.92 c,d 34.62 ± 0.49 a 27.90 ± 5.15 a,b 10.89 ± 1.81 d,e,f 12.42 ± 0.19 c,d,e 19.71 ± 1.79 b,c 11.23 ± 2.23 e 15.31 ± 3.45 c,d,e 16.89 ± 1.78 c,d,e 14.08 ± 2.15 c,d,e 11.99 ± 2.73 d,e

Values are mean ± SD values (in nmol/µmol of phennylalanine) of, at least, three independent experiments. Means with the same letters (b-g) in the same column are not significantly different (p < 0.05). Abbreviations: DD, 2,4-decadienal; ED, 4,5-epoxy-2-decenal; EH, 4,5-epoxy-2-heptenal; HD, 2,4-heptadienal; HN, 4,-hydroxy-2-nonenal; LnOOMe, methyl 13-hydroperoxyoctadeca-9,11,15-trienoate; LOOH, 13-hydroperoxyoctadeca-9,11-dienoic acid; LOOMe, methyl 13-hydroperoxyoctadeca-9,11-dienoate; OC, 2-octenal; OH, 4-oxo-2-hexenal; ON, 4-oxo-2-nonenal; Phe, phenylalanine

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Figure 1



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TOC Graphics


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