Linoleic acid hydroperoxide reacts with hypochlorous acid, generating peroxyl radical intermediates and singlet molecular oxygen

Abstract

The reaction of hypochlorous acid (HOCl) with hydrogen peroxide is known to generate stoichiometric amounts of singlet molecular oxygen [O2 (1Deltag)]. This study shows that HOCl can also react with linoleic acid hydroperoxide (LAOOH), generating O2 (1Deltag) with a yield of 13 +/- 2% at physiological pH. Characteristic light emission at 1,270 nm, corresponding to O2 (1Deltag) monomolecular decay, was observed when HOCl was reacted with LAOOH or with liposomes containing phosphatidylcholine hydroperoxides, but not with cumene hydroperoxide or tert-butyl hydroperoxide. The generation of O2 (1Deltag) was confirmed by the acquisition of the spectrum of the light emitted in the near-infrared region showing a band with maximum intensity at 1,270 nm and by the observation of the enhancing effect of deuterium oxide and the quenching effect of sodium azide. Mechanistic studies using 18O-labeled linoleic acid hydroperoxide (LA18O18OH) showed that its reaction with HOCl yields 18O-labeled O2 (1Deltag) [18O2 (1Deltag)], demonstrating that the oxygen atoms in O2 (1Deltag) are derived from the hydroperoxide group. Direct analysis of radical intermediates in the reaction of LAOOH with HOCl by continuous-flow electron paramagnetic resonance spectroscopy showed a doublet signal with a g-value of 2.014 and a hyperfine coupling constant from the alpha-hydrogen of a(H) = 4.3 G, indicating the formation of peroxyl radicals. Taken together, our results clearly demonstrate that HOCl reacts with biologically relevant lipid hydroperoxides, generating O2 (1Deltag). In addition, the detection of 18O2 (1Deltag) and peroxyl radicals strongly supports the involvement of a Russell mechanism in the generation of O2 (1Deltag).

Fig. 1.

Monomol light emission of O₂ (¹Δ_g) generated in the reaction of HOCl with different hydroperoxides. Light emission observed upon injection of 0.17 ml of 10 mM HOCl (final concentrationm, 1 mM) into 1.5 ml of 1 mM H₂O₂ (A), 1 mM LAOOH (B), 1 mM t-BuOOH (C), and 1 mM CuOOH (D).

Fig. 2.

Monomol light-emission spectrum of O₂ (¹Δ_g) generated in the reaction of HOCl with LAOOH (A) and H₂O₂ (B).

Fig. 3.

Effect of pH on O₂ (¹Δ_g) generation in the reaction of LAOOH with HOCl. Reaction was initiated by injection of 0.17 ml of 10 mM HOCl (final concentration, 1 mM) into 1.5 ml of 1 mM LAOOH solution in 0.1 M phosphate buffer prepared in D₂O at the following pHs 6.0, 7.0, 7.4, 8.0, and 9.0.

Fig. 4.

Monomol light emission of O₂ (¹Δ_g) observed during injection of HOCl into PC liposomes containing different concentrations of PCOOH. (A) Light emission monitored during injection of 0.17 ml of 100 mM HOCl (final concentration 10 mM) into 1.5 ml of 1 mM PC vesicles containing 0, 10, 20, or 50% PCOOH. (B) The amount of O₂ (¹Δ_g) calculated by integration of the area under emission signal. The percentages indicate the yield of O₂ (¹Δ_g).

Fig. 5.

Analysis of EAS endoperoxides formed in the reaction of LA¹⁸O¹⁸OH (5 mM) with HOCl (10 mM) in the presence of EAS (8 mM) by HPLC-MS/MS. (A) HPLC chromatogram monitored at UV 215 nm. Mass chromatograms obtained by selecting the ions at m/z 228 (B), m/z 229 (C), and 230 (D).

Figure 6

Fig. 6.

Electrospray ionization (ESI)-MS spectrum of the EAS endoperoxides formed by the reaction of LA^xO^xOH (x = 16 or 18) with HOCl. (A) LA¹⁶O¹⁶OH (5 mM) was reacted with 10 mM HOCl in 0.1 M phosphate buffer, pH 7.4. (B) LA¹⁸O¹⁸OH (5 mM) was reacted with 10 mM HOCl in 0.1 M phosphate buffer, pH 7.4 in 85% D₂O, 10% H₂O, and 5% MeOH. (C) Reaction described in B without oxygen.

Fig. 7.

EPR spectrum of linoleate peroxyl radicals. (A) Experimental spectrum obtained during the reaction of HOCl with LAOOH. Spectrometer settings were microwave frequency, 9.650 GHz; microwave power, 10 mW; field-modulation frequency, 100 kHz; field-modulation amplitude, 3 G; receiver gain, 1 × 10⁶; time constant, 164 ms; scan rate, 1.2 G s-1; number of scans, 1. (B) Computer simulation of spectrum A using the following values: aH = 4.3 G; line width, 2.8 G; center of the spectrum, 3,423 G.

Figure 9