Methods - Free-Radical-Induced DNA Damage and Its Repair - page 473

their determination (Dizdaroglu 1985, 1990, 1991, 1992; Dizdaroglu and Bergtold
1986; Fuciarelli et al. 1989; Dizdaroglu and Gajewski 1990; Jaruga et al. 2001).

13. 2 . 4
Determination of DNA Lesions by Liquid Chromatography

When the term 'liquid chromatography' is used these days, it usually refers to
HPLC (for reviews see Zakaria and Brown 1981; Scoble and Brown 1983), but
valuable separations can also be achieved by TLC (Cadet et al. 1983). Although
detection in HPLC is mainly by UV spectrophotometry ( e.g., Cadet et al. 1982),
electrochemical detection is the method of choice in the case of the strongly re-
ducing 8-oxo-G lesion (Floyd et al. 1986; Shigenaga et al. 1989, 1990; Berger et
al. 1990; Mei et al. 2003). In a multi-laboratory test, this method proved to be
much more reliable than GC/MS and HPLC/MS-MS methods (ESCODD 2003).
The work-up of biological samples and the difficulties that one might encounter
have been discussed (Claycamp and Ho 1993; Douki et al. 1997; Dany et al. 1999).
This is of importance, since this compound is often used as a marker of oxidative
DNA damage, for example, in assessing the role of dietary fats (Loft et al. 1998).
Electrochemical detection is also the method of choice for the reducing
dCyd-derived products 5OHdCyd and 5OHdUrd that can be released enzymati-
cally from oxidized DNA (Wagner et al. 2004). After water elimination, the Ug
may also be determined by this technique.
The sugar lesions FUR and 5-MF (Joshi and Ganesh 1994), as well as DNA-ad-
ducts (Park et al. 1989) have also be detected with the help of HPLC.
For optical detection, the product to be determined must have a reasonable
absorption coefficient. Saturation of the pyrimidine C (5) C (6) bond destroys the

chromophore, and for the determination of Tg yields excreted into the urine,
this product was reduced with HI into Thy prior to an HPLC analysis (Cathcart
et al. 1984).
The combination of HPLC and MS (cf. Frelon et al. 2000) is expected to be-
come the method of choice in many cases. The state of the art has been reviewed
(Cadet et al. 2002).

−

13. 2 . 5
MALDI-TOF

Another very sensitive MS technique is MALDI-TOF. Its high-resolution mass
spectra allowed, for example, the identification of the 8-oxo-G lesion by its +16-
Da mass shift in a oligonucleotide heptamer at picomol level (Bartolini and
Johnston 2000).

13. 2 .6
Capillary and Pulsed Field Gel Electrophoresis

Capillary gel electrophoresis (CGE) has the advantage of a short analysis time,
high degree of automation and reproducibility combined with a good resolving
power for dsDNA fragments. The mobility of a DNA fragment is given by equa-