protein is estimated based on the residual radioactivity in the proteins. An
example of covalent protein binding studies in liver and plasma of male rats
orally dosed with the tritium-labeled dihydrobenzoxazhiin analogEat 20 mg/
kg is shown in Table 14.2. The covalent protein binding of AE was
below 20 pmol-equiv./mg protein in rat liver and plasma samples. All
procedures on these animals were in accordance to established guidelines and
were reviewed and approved by the Institutional Animal Care and Use
Committee (IACUC).
14.2.5.2 Evaluation of the Loss of the^3 H-Label in Rat Urine Urine samples
of rats (n= 3) at 24 h time point are pooled and centrifuged for 10 min. A
portion of urine sample (3 mL) is taken, and 10mL of the sample is counted for
radioactivity. The remaining sample is loaded onto a preconditioned 3 cm3cc
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 radioactivity. The second
portion (0.5 mL) of the last eluate is evaporated to dryness under nitrogen
overnight. The residue is reconstituted in 0.5 mL of MeOH–H 2 O (2:1) and is
counted for radioactivity. The difference in radioactivity between prior- and
after evaporation samples is the loss of the amount of the tritium label. By
dividing the amount of the lost tritium by the radioactivity in the original urine
sample, an extent of tritium loss for the urine sample is estimated (Note 4).
14.2.6 Notes
(1) For the optimal performance of retaining drug-related components on
the solid phase extraction columns, samples should be passed through
the columns by gravity. However, if needed, very low vacuum may be
applied.
(2) Additional incubations may also be conducted in the presence of sodium
cyanide (1 mM) or glutathione (5 mM) for 60 min.
(3) Readers may also refer to the published procedures for measuring the
covalent protein binding using this semiautomated method (Day et al.,
2005).TABLE 14.2 Covalent protein binding of [^3 H]E in liver and plasma of rats.a
Covalent protein binding (pmol equiv./mg protein)Tissue 2 h 6 h 24 h
Liver 7 116 22 2
Plasma 1 0.2 2 0.2 2 0.2
aMale rats were orally dosed with [ (^3) H]E at 20 mg/kg. Three rats per time point.
466 PROTOCOLS FOR ASSESSMENT