Vitamin C-Induced Decomposition of Lipid Hydroperoxides to Endogenous Genotoxins
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J. Cadet et al. Mutat. Res. 424 9 (1999).
Johnson T. M., Yu Z.-X., Ferrans V. J., Lowenstein R. A., Finkel T., Proc. Natl. Acad. Sci. U.S.A. 93, 11848 (1996).
Mass spectrometry was conducted with a Thermo Finnigan LCQ ion trap mass spectrometer equipped with an APCI source. The mass spectrometer was operated in the positive ion mode with a discharge current of 5 μA applied to the corona needle. Nitrogen was used as the sheath (80 psi) and auxiliary (10 units) gas. The vaporizer and heated capillary temperatures were set at 450°C and 150°C respectively. Full scanning analyses were performed in the range of m/z 100 to m/z 800. Collision-induced dissociation experiments coupled with MS/MS analysis used helium as the collision gas. The relative collision energy was set at 20% of maximum (1 V). Ionization of the polar 4-hydroxy-2-nonenal was more efficient than the ionization of the other bifunctional electrophiles so the mass spectrometer response was almost an order of magnitude greater.
For normal-phase LC system 1 we used two silica columns in series (250 mm by 4.6 mm internal diameter 5 μm) with a post-column split of 160 μl/min to the UV detector (226 nm) and 640 μl/min to the mass spectrometer. Solvent A was hexane/2- propanol (197:3 v/v) and solvent B was hexane/2-propanol (70:30 v/v). Isocratic elution with 21% B was conducted for 13 min at a flow rate of 800 μl/min. A linear gradient was then run to 25% B for 18 min. For normal-phase LC system 2 we used only one of the silica columns and no split to the UV detector (226 nm). Isocratic elution with 3% B was conducted for 15 min at 1.0 ml/min. Retention times were as follows: trans -4 5-epoxy-2( E )-decenal (4.9 min) 4-oxo-2-nonenal (5.1 min) cis -4 5-epoxy-2( E )-decenal (5.5 min) 4-hydroperoxy-2-nonenal (7.5 min) 4-hydroxy-2-nonenal (12.3 min). For LC/MS experiments 13-HPODE (800 nmol 400 μM) was allowed to decompose in the presence of 200 nmol of vit C (100 μM) or 2000 nmol of VIt C (1 mM) in 2 ml of pH 7.0 Chelex-treated Mops buffer (100 mM) containing NaCl (150 mM) (14) at pH 7.0 and 37°C. Aldehydes were extracted with diethyl ether (0.5 ml) and an aliquot (20 μl) was analyzed by LC/APCI/MS with concomitant UV monitoring with system 1. For quantitation experiments 13-HPODE (80 nmol 400 μM) in 200 μl of Chelex-treated pH 7.0 Mops buffer (100 mM) containing NaCl (150 mM) was reacted with increasing amounts of vit C for 2 hours at 37°C. Aldehydes were extracted into diethyl ether (200 μl) and an aliquot (25 μl) was analyzed by LC/UV with system 2. Quantitation was performed from standard curves constructed by quantitation of known amounts of authentic standards extracted from Mops buffer with ether.
Authentic 4-oxo-2-nonenal was prepared as described in (15) authentic trans -4 5-epoxy-2( E )-decenal and cis -4 5-epoxy-2( E )-decenal were prepared as in (21) authentic 4-hydroperoxy-2-nonenal was prepared enzymatically from 3( Z )-nonenal as described in (22) and 4-hydroxy-2-nonenal was obtained from Cayman (Ann Arbor MI). All samples were >98.5% pure as determined by normal-phase LC with system 2.
Levine R. L., et al., Cancer Res. 60, 4098 (2000).
F.-L.Chung
We thank Thermo Finnigan (San Jose CA) for the kind gift of an APCI source and G. A. FitzGerald T. M. Penning and L. J. Marnett for helpful discussions. Supported by NIH grants CA65878 and CA16520.