Methods - Free-Radical-Induced DNA Damage and Its Repair - page 475

expression and is repaired by the nucleotide excision repair pathway (Brooks et al.
2000) has also been developed for the use in mammalian tissues (Randerath et al.
2001). Ade- N -1-oxide, a product that is formed when DNA is exposed to H 2 O 2 , has
also been determined using this technique (Mouret et al. 1990).

13. 2 . 8
Detection of Hydroperoxides

Hydrogen peroxide and organic hydroperoxides can be reduced by molybdate-
activated iodide [Allen et al. 1952; reactions (13) and (14)]. The iodine atoms
formed in these reactions combine to iodine which complexes with iodide [reac-
tions (15) and (16)], and it is this I 3 − complex which is measured ( ε (3540 nm) =

25,000 dm 3 mol −1 cm −1 )

ROOH + I − RO • + I • + OH − → (13)

RO • + I − RO − + I •

→ (14)

2 I •

→ I 2 (15)

I 2 + I − I 3 −

→ (16)

Hydrogen peroxide reacts considerably faster with this reagent (t 1/2 = 2.5 s) than
organic hydroperoxides, and the differences in the rate of buildup not only al-
lows to determine H 2 O 2 and organic hydroperoxides side by side, but often the
rate of reaction of a given hydroperoxide with this reagent can be used as a fin-
gerprint for its presence (Dowideit and von Sonntag 1998; Flyunt et al. 2003). A
certain disadvantage of this reagent is the fact that the iodine thus formed may
react with other products (Al-Sheikhly et al. 1984). This problem can be over-
come in certain cases by combining it with an HPLC separation (post-column
derivatization mode). For a quantification of a hydroperoxidic product by this
method the slowness of reaction of some hydroperoxides with this reagent may
require the heating of the reaction coil, but in unfavorable cases the reaction
may still remain incomplete. This may result in an underestimate of the hydro-
peroxide yield.
Hydroperoxides also react readily with Fe 2+ in acid media [reactions (17)
and (18)]. The Fe 3+ formed can be complexed with xylenol orange. This complex
has a strong absorption coefficient in the visible (

(540 nm) = 26,800 dm 3 mol −1
cm −1 ). This method has been used with some advantage in the identification of
the hydroperoxides formed upon • OH-attack on Thd by HPLC using the post-
column derivatization technique (reagent: e.g. 2.3

ε

10 −4 mol dm −3 ammonium ×

10 −2 mol dm −3 sulfuric acid, 5.6 10 −4 mol dm −3 xylenol ferrous sulfate, 3.5 × ×

orange; Wagner et al. 1990).

ROOH + Fe 2+ RO • + OH − + Fe 3+

→ (17)

RO • + Fe 2+ RO − + Fe 3+

(18)

→