Functional in vivo EPR Spectroscopy and Imaging Using Nitroxide and Trityl Radicals - Stable Radicals: Fundamentals and Applied Aspects of Odd-Electron Compounds - page 560

(a)

0.0 0.1

Rate constant (min − 1 )

(b)

Figure 16.16 Redox mapping of tumor of untreated (a) and BSO-treated (b) mice. 2-D spatial mapping of
pseudo-firstorderrateconstantsofthenitroxide3-CPreductionobtainedinRIF-1tumorsimplantedintheupper
legofamouseandmeasuredusinginvivoL-bandEPRimaging. 128 Therateofreductionwassignificantlyslower
in tumors of mice treated for 6h with 2.25mmol/kg of L-buthionine-S,R-sulfoximine (BSO), a GSH depleting
agent.(Reprintedfrom[17],Copyright2002,AmericanAssociationforCancerResearch.)

The use of fluorometric, photometric, and chromatographic assays for GSH measurements is mostly lim-
ited to in vitro or ex vivo systems, due to the invasiveness involved and the limited depth of penetration of
light. 133 - 136 NMR based approaches normally detect endogenous GSH but have somewhat low sensitivity
and the spectral assignment is complicated due to the overlapping of numerous resonances. 137 - 140
EPR spectroscopy in combination with thiol-specific nitroxides allows determination of the accessible
thiol groups in various biological macromolecules, such as human plasma low density lipoproteins 141 and
erythrocyte membranes. 142 This approach normally requires purification of the sample from the unbound
label and cannot be used in vivo . Moreover, application of thiol-specific mononitroxides for the EPR
measurement of glutathione or cysteine is hardly possible due to insignificant EPR spectral changes of
the label upon binding to low molecular weight compounds. The latter limitation was overcome by the
development of the disulfide nitroxyl biradicals, DNB 12,143,144 (Scheme 16.9). The principal advantage of
the DNB reagents is the large EPR spectral changes that accompany their reaction with low molecular
weight thiols such as GSH (Figure 16.17).