Food Protein Sterylation: Chemical Reactions between Reactive Amino Acids and Sterol Oxidation Products under Food Processing Conditions

Abstract

Sterols, especially cholesterol and phytosterols, are important components of food lipids. During food processing, such as heating, sterols, like unsaturated fatty acids, can be oxidized. Protein modification by secondary products of lipid peroxidation has recently been demonstrated in food through a process called lipation. Similarly, this study was performed to assess, for the first time, the possibility of reactions between food proteins and sterol oxidation products in conditions relevant for food processing. Therefore, reaction models consisting of oxysterol (cholesterol 5α,6α-epoxide) and reactive amino acids (arginine, lysine, and methionine) were incubated in various conditions of concentration (0-8 mM), time (0-120 min), and temperature (30-180 °C). The identification of lysine adducts through thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) with a diode array detector (DAD), and electrospray ionization (ESI) mass spectrometry (MS) evidenced a reaction with lysine. Moreover, the HPLC-ESI with tandem mass spectrometry (MS/MS) analyses allowed observation of the compound, whose mass to charge ratio m/z 710.5 and fragmentation patterns corresponded to the reaction product [M + H]⁺ between cholesterol-5α,6α-epoxide and the ε-amino-group of N_α-benzoylglycyl-l-lysine. Moreover, kinetic studies between N_α-benzoylglycyl-l-lysine as a model for protein-bound lysine and cholesterol 5α,6α-epoxide were performed, showing that the formation of lysine adducts strongly increases with time, temperature, and oxysterol level. This preliminary study suggests that in conditions commonly reached during food processing, sterol oxidation products could react covalently with protein-bound lysine, causing protein modifications.

Keywords: amino acids; cholesterol; lipation; lysine; oxyphytosterols; oxysterols; phytosterols; protein modification; sterylation.

Figure 1

Chemical structures of cholesterol, principal plant sterols, and oxysterols.

Figure 2

Overlaid chromatograms of N_α-benzoylglycyl-l-lysine and derivatives in the blank sample (blue) and the reaction mixture (green) incubated in the same conditions (60 min, 160 °C). In the mixture, there is the formation of a reaction product (retention time R.T.: 11.7 min).

Figure 3

Proposed chemical reactions: (a) cholesterol 5α,6α-epoxide and N_α-Boc-L-lysine; (b) cholesterol 5α,6α-epoxide and N_α-benzoylglycyl-l-lysine. E.M: exact mass (g/mol).

Figure 4

Molecular ions and respective mass spectra of products from reaction mixtures between (a) cholesterol 5α,6α-epoxide and N_α-Boc-l-lysine; (b) cholesterol 5α,6α-epoxide and N_α-benzoylglycyl-l-lysine. E.I.C: Extract ion chromatogram. ESI: Electrospray ionization.

Figure 5

Mass spectrometry (MS) spectra recorded during high-performance liquid chromatography (HPLC)-MS/MS measurements at the retention time of the reaction product between cholesterol-5α,6α-epoxide and N_α-benzoylglycyl-l-lysine (m/z 710.5). (A) HPLC-MS/MS measurements of (a) cholesterol-5α,6α-epoxide, (b) N_α-benzoylglycyl-l-lysine, and (c) a mixture of cholesterol-5α,6α-epoxide and N_α-benzoylglycyl-l-lysine, each heated at 180 °C for 120 min; (B) product ion spectrum (parent ion, m/z = 710.5 ± 0.5; fragmentor voltage, 135 V; collision energy, 30 V) of the reaction product from the mixture of cholesterol-5α,6α-epoxide and N_α-benzoylglycyl-l-lysine that had been heated at 180 °C for 120 min.

Figure 6

Overlaid chromatograms of N_α-benzoylglycyl-l-lysine and derivatives in blank samples after different times of incubation at 180 °C (T1: 5 min, T2: 10 min, T3: 30 min, and T4: 60 min).

Figure 7

Overlaid chromatograms of N_α-benzoylglycyl-l-lysine and derivatives in mixture reaction samples after different times of incubation at 180 °C (T1: 5 min, T2: 10 min, T3: 30 min, and T4: 60 min).

Figure 8

Kinetic studies: evolution of N_α-benzoylglycyl-l-lysine levels in the reaction mixture (orange) and blank samples (blue). (a) Effect of time on the reaction between cholesterol 5α,6α-epoxide and the ε-amino-group of lysine (incubation temperature: 180 °C). (b) Effect of temperature on the reaction between cholesterol 5α,6α-epoxide and the ε-amino-group of lysine (incubation time: 120 min).

Figure 9

Kinetic studies: evolution of the reaction product levels from the reaction between N_α-benzoylglycyl-l-lysine and cholesterol 5α,6α-epoxide. (a) Effect of time on the reaction between cholesterol 5α,6α-epoxide and the ε-amino-group of lysine (incubation temperature: 180 °C). (b) Effect of temperature on the reaction between cholesterol 5α,6α-epoxide and the ε-amino-group of lysine (incubation time:120 min). (c) Effect of oxysterol concentration (incubation at 180 °C for 120 min). Epoxide/Lysine = concentration of cholesterol 5α,6α-epoxide/concentration of N_α-benzoylglycyl-l-lysine.