GSTs in liver (GSTA, GSTM, GSTP) can be prepared by affinity
chromatography on S-linked glutathione agarose (Sigma G4510) or immo-
bilizedS-hexylglutathione. After loading of the cytosol and washing with
0.15MNaClina0.04Mphosphatebuffer,pH7.0,theGSTscanbeeluted
with 50 mM GSH in a 0.1 M Tris buffer, pH 9.6 (Coles and Kadlubar, 2005).
Hepatocytes may also be used, but depending on the cell–cell contact and
transporter function, excretion of the conjugates out of the cells may not
occur, thus requiring lysis of the cells for measurement. In cases where the cells
are well polarized (e.g., hepatocyte spheroids), significant canalicular
transport can occur resulting in the appearance of glutathionyl and
cysteinyl–glycine conjugates. It has been reported that cryopreserved
hepatocytes have low glutathione concentrations upon thawing and may
lead to underreporting of GSH conjugate production compared to freshly
isolated cells (Sohlenius-Sternbeck and Schmidt, 2005). Cloned, expressed
GSTs (GSTA1-1, GSTM1-1, and GSTP1-1 homodimers) are also commer-
cially available (Panvera/InVitrogen or Sigma) and can be used to determine
the activity of individual enzymes or for detailed kinetic studies with
individual substrates. With purified enzymes or cloned, expressed prepara-
tions glutathione is added. Typical GSH concentrations range from 0.3 to
5mM.TheKmfor GSTA1-1 has been reported as 120mM for CDNB and with
rat GSTA1-1 (Schramm et al., 1984) theKmwas 0.16 mM for CDNB and
0.81 mM for trans-4-phenyl-3-buten-2-one (Warholm et al., 1983).
Intracellular GSH concentrations vary depending upon the tissue but in
hepatocytes and leukocytes the concentration is5 mM. However, at high
GSH and substrate concentrations, nonenzymatic formation of GS-conjugates
may occur. A variety of buffers have been used: Tris buffers at pH 7.5 are
commonly employed but pH optima may vary from 6.5 to 8. Tris-maleate buffers
can extend the buffering capacity to cover lower pHs if desired.
Glutathione conjugates are highly polar, ionized conjugates that are
generally analyzed by either HPLC or LC/MS methods. In organic/water
mobile phases, the conjugates will elute in the solvent front. In acidic mobile
phases containing acetic acid or trifluoroacetic acid (TFA), the amino acid
carboxyl groups are unionized, and the protonated amino groups can form ion
pairs with the organic acids, resulting in significant retention. In the case of
acetaminophen, we have found that both the glutathione conjugate and
acetaminophen elute after acetaminophen in the presence of 0.05% TFA in the
mobile phase. TFA is commonly used in peptide analysis, but can cause some
ion suppression in electrospray mass spectrometry, particularly in the negative
ion mode. In the aforementioned article by Stern et al., acetaminophen and its
glutathione-derived conjugates were analyzed by atmospheric pressure positive
ion MS (Stern et al., 2005). While fragmentation patterns can be quite complex,
typical fragmentation of peptide bonds is generally observed and easy to follow
especially with an ion trap mass spectrometer, where the bonds that are easiest
to break can be followed down in an MSnanalysis (e.g., neutral loss of the
gamma-glutamyl (M-130) and glycine moieties from glutathione).
82 CONJUGATIVE METABOLISM OF DRUGS