Formation of the toxic furan metabolite 2-butene-1,4-dial through hemin-induced degradation of 2,4-alkadienals in fried foods

Abstract

Background: The mechanism of protein modification by 2,4-alkadienals (ADE), lipid peroxidation products prevalent in fried foods, was investigated through model reactions.

Results: A mixture of 2,4-heptadienal (HDE) and hemin was initially incubated at pH 3.0-7.4, followed by treatment with acetyl-cysteine (AcCys) and acetyl-lysine (AcLys) at pH 7.4. Analysis via HPLC revealed a product with a characteristic UV spectrum as the primary peak. This product was identified as an AcCys-pyrrole-AcLys (CPL) crosslink derived from AcCys, 2-butene-1,4-dial (BDA), and AcLys. Increasing the HDE concentration in the initial reaction led to maximum CPL formation at pH 3.5 in the presence of hemin. Lowering the HDE concentration with a higher Cys/HDE ratio resulted in CPL formation, which was observed at pH 7.4 and 3.5 in the presence of hemin. Upon incubation of ADE and hemin at pH 3.0-3.5, BDA was directly identified as 2,4-dinitrophenylhydrazone. BDA was also detected in the 2,4-decadienal reaction mixture. Additionally, a notable propensity for high BDA-dC adduct formation with hemin under acidic conditions was observed, consistent with the results of CPL assay and BDA-2,4-dinitrophenylhydrazone analysis.

Conclusions: 1) BDA is efficiently generated from ADE in the presence of hemin under gastric conditions, and 2) BDA-derived CPL can also form under physiological conditions (pH 7.4) through the interaction of ADE, hemin, Cys, and Lys. BDA is recognized as the primary reactive metabolite of the suspected carcinogen furan (IARC, 2B). Given that human intake of ADE exceeds that of furan and acrylamide (IARC 2A) by several orders of magnitude, and the estimated hemin concentration in the stomach post-meal is comparable to the present study, a substantial amount of BDA may form in the stomach following consumption of fried foods and meat. The risk assessment of ADE warrants a thorough re-evaluation, based on the toxicity mechanism of BDA.

Keywords: 2,4-decadienal; 2,4-heptadienal; 2-butene-1,4-dial; Cysteine-pyrrole-lysine; Fried foods; Furan; Protein crosslink.

Fig. 1

Isolation and identification of AcCys-pyrrole-AcLys (CPL) in the reaction mixture of (hemin, HDE, pH 4.5), followed by the addition of AcCys and AcLys. a Analyses of the reaction mixture and (b) synthetic CPL. The upper figures display the UV spectra of peaks a’ and b’

Fig. 2

Mass spectra of the primary product a’ in (a) Mass chromatogram (negative TIC). b Mass spectrum (positive). c Mass spectrum (negative)

Fig. 3

Summary of the formation of CPL using different concentrations of HDE: a high HDE concentration (Method B), b low HDE concentration (Method C), and (c) low HDE concentration without additives (Method D). d Time course of CPL formation using Method D, with data points for pH 7.4 (●), pH 3.5 (〇), no data (NO), and not detected (nd). The blue bar denotes the peak area. The orange bar indicates the formation rate [peak area / initial HDE concentration (mM)]

Fig. 4

Detection of BDA as 2,4-dinitrophenylhydrazone: a analysis of authentic BDA–DNPH, b analysis of BDA formed in model reactions. The upper figures display UV spectra of peaks a’ and b’

Fig. 5

Mass spectra of BDA–DNPH obtained from reactions of HDE and DDE, and from standard BDA

Fig. 6

Formation of BDA from ADE under various conditions analyzed using the DNPH method. △, HDE, pH 3.5, + hemin; □, HDE, pH 3.5, -hemin; 〇, DDE, pH 3.0, + hemin; ●, HDE, pH 3.0, + hemin; ■, HDE, pH 3.0, + hemin, + tBuOOH; ▲, HDE, pH 7.4, + hemin; ×, HDE, pH 7.4, -hemin; Inset: calibration curve plotting BDA concentration on the horizontal axis and the peak area of BDA–DNPH on the vertical axis

Fig. 7

Summary of BDA-dC adduct formation: a reaction involving high concentrations of HDE (mixture B + dC); b reaction involving low concentrations of HDE (mixture C + dC); NO, no data. The blue bar denotes the peak area. The orange bar indicates the formation rate [peak area / initial HDE concentration (mM)]

Fig. 8

Formation of CPL and dC adducts from ADE via BDA

Fig. 9

Summary and speculations