The Hydroxyl Radical - Free-Radical-Induced DNA Damage and Its Repair - page 55

be of importance in the in vivo production of • OH. It has, therefore, been sug-
gested (Acworth et al. 1999) to reduce the salicylate concentration, not realizing
that a reduction of the probe concentration also reduces its scavenging capacity
(Eq. 43).
Some systems that have been proposed as suitable • OH probes (for reviews
see Hageman et al. 1992; Kaur and Halliwell 1994, 1996; Loft and Poulsen 1999;
von Sonntag et al. 2000), and the principles on which they are based (and, if pos-
sible, their reliability) will be discussed in the following.
3.5.1
Aromatic Hydroxylation
Aromatic hydroxylation is most commonly used for the detection of • OH. How-
ever, the primary • OH adducts must be oxidized to yield the final product(s).
Disproportionation reactions produce these compounds usually only in very low
yields. For this reason, an oxidant is required. Although oxygen may serve as an
oxidant, the yields are not quantitative because of side reactions (Chap. 8). The
addition of a one-electron oxidant, for example Fe(CN) 6 3− , may overcome this
problem (Volkert and Schulte-Frohlinde 1968; Bhatia and Schuler 1974; Madha-
van and Schuler 1980; Buxton et al. 1986), but in certain cases an even stronger
oxidant such as IrCl 6 2− may be required (Fang et al. 1996).
Quite a number of systems that might give rise to • OH-typical products, that
is, products that are not formed in enzymatic oxidation processes, have been
proposed. For example, phenylalanine (present in all proteins and thus can
serve as an internal marker; Karam et al. 1984; Karam and Simic 1988a,b; Kaur
et al. 1997), tyrosine (Maskos et al. 1992), terephthalic acid (Armstrong et al.
1963; Matthews 1980), salicylic acid (Ingelmann-Sundberg et al. 1991; Coudray
et al. 1995; Bailey et al. 1997) and its isomer 4-hyroxybenzoic acid (Ste-Marie et
al. 1996), 5-aminosalicylic acid (Kumarathasan et al. 2001), dopamine (Slivka
and Cohen 1985), phthalic hydrazide (Backa et al. 1997), coumarin-3-carboxylic
acid (Makrigiorgos et al. 1993, 1995; Chakrabarti et al. 1996, 1998; Manevich et
al. 1997; Parker 1998), antipyrine (Coolen et al. 1997) and CO 2 formation as a
by-product of the hydroxylation of benzoic acid [Lamrini et al. 1994; reaction
(10)]. The very sensitive luminescence detection methods, commonly used in
vitro, are not feasible in vivo (Hirayama and Yida 1997). Some of the systems,
especially mechanistic implications, will be discussed below in more detail.
It was mentioned above that in aromatic hydroxylation an oxidant is required,
and the product yields vary considerably with the oxidant used (for the reason
why O 2 does not serve as a typical one-electron oxidant, see Chap. 8). A typi-
cal example is the formation of tyrosines from phenylalanine (Table 3.4). Their
yields are especially low in the absence of an oxidant, since dimerization usually
dominates over disproportionation in these systems. The determination of the
products is usually done by either HPLC or GC/MS after trimethylsilylation, and
the proteins have to be hydrolyzed prior to analysis. Attention has been drawn
to the fact that in vivo cytochrome P-450 enzymes hydroxylate phenylalanine to
p -tyrosine (Bailey et al. 1997).