Oxidative versus Non-oxidative Decarboxylation of Amino Acids

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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

2

phenylacetaldehyde as a consequence of phenylalanine degradation by carbonyl

3

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

4

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

5

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

6

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

7

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

8

phenylethylamine/phenylacetaldehyde ratio depended on both the carbonyls and the

9

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

10

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

11

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

12

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

13

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

14

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

15

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

16

favored.

17 18

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

201

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

212

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

213

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

233

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

235

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

236

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

237

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

238

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

239

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

241

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

243

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

246

aldehyde.

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

248

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

249

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

250

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

251

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,

254

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

255

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

256

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

257

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

258

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

259

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

260

phenylacetaldehyde. Benzaldehyde formation also decreased when amount of water

261

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-

263

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

264

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

265

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

266

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

267

increased.


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

269

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

272

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

274

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

275

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

276

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

277

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

278

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

279

assayed temperatures were calculated using the equation

280

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

281

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

282

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

283

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

284

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

285

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 >

287

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

288

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

289

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

291

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

293

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

295

6.

296

Differently to 2-phenylethylamine and phenylacetaldehyde, benzaldehyde was

297

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

298

determine its formation Ea.

299

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

300

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

301

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

302

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

303

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

304

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

305

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

306

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

308

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

309

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

310

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

311

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

317

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

318

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

20

ACS Paragon Plus Environment

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Figure 1



A

8 PEA/PAC ratio

PEA/PAC ratio

6

4

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B

6 4 2

0 0

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Figure 2


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1 0 -1 -2 -3 0.0022

0.0024

0.0026 -1

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Figure 3



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Figure 4


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TOC Graphics


Oxidative versus Non-oxidative Decarboxylation of Amino Acids

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