was reported that the covalent protein binding values of reference model
compounds in human and rat liver microsomes were136 and 574 pmol-
equiv./mg protein for [^3 H]imipramine, 68 and 413 pmol-equiv./mg protein for
[^14 C]diclofenac, 1405 and 1578 pmol-equiv./mg protein for [^14 C]naphthalene,
respectively (Day et al., 2005). An example of covalent protein binding studies
of the tritium-labeled dihydrobenzoxazhiin analogEin human liver micro-
somes is shown in Fig. 14.6. The covalent protein binding ofEwas both
NADPH and time dependent. In the presence of NADPH, the binding was
685 pmol-equiv./mg protein and was attenuated significantly by glutathione or
potassium cyanide (Fig. 14.6).
14.2.2.2 Evaluation of the Loss of the^3 H-Label in Incubations of^3 H-Tracers
with Human or Rat Liver Microsomes Incubations of^3 H-tracers with human
or rat liver microsomes are performed for 60 min under the same conditions as
described above. Final incubation volume is 1 mL. Reactions are terminated
by adding 100mL of 10% trifluoroacetic acid (TFA) to the final TFA
concentration of 1%. Water (400mL) is then added to the above mixture,
and the resulting sample is sonicated for 10 min and vortexed for 10 min.
After centrifugation at 2500 gfor 20 min, a portion of supernatant (10mL) is
counted for radioactivity. The remaining sample is loaded on a preconditioned
3 cmcc Oasis HLB column (prewashed with water, MeOH, and water), and
is slowly passed through the column by gravity (Note 1). The eluate is vortexed
and loaded onto a second preconditioned Oasis HLB column. The collected
eluate is then vortexed and loaded onto a third preconditioned Oasis
HLB column. One portion (0.5 mL) of the last eluate is counted for
0 min 30 min 60 min 60 min 60 min 60 min
+NADPH +NADPH +NADPH +NADPH +NADPH -NADPH
+GSH +KCN6004002000pmol equiv. 1/mg proteinFIGURE 14.6 Covalent protein binding of [^3 H]Ein human liver microsomes in the
presence or absence of NADPH, glutathione and sodium cyanide. Incubations were
performed, as described in Section 14.1.2.
PROTOCOLS FORIN VITROANDIN VIVOCOVALENT PROTEIN 463