Clinical Biochemistry of Domestic Animals (Sixth Edition) - UMK ...

IV. Mature RBC
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released during Hb oxidative denaturation, thereby reducing
the potential of hemin for membrane injury ( Shviro
and Shaklai, 1987 ). GSH functions as an electron donor in
various reductive enzyme reactions including glutathione
peroxidase (GPX), phospholipid hydroperoxide glutathione
peroxidase, glutathione S-transferase, and glutaredoxin
(discussed subsequently).
Although GSH is constantly being oxidized to a disulfide
(GSSG) within RBCs, most glutathione is maintained in its
reduced form by the glutathione reductase reaction. RBCs
increase PPP metabolism to provide the NADPH necessary
for the regeneration of GSH by the GR reaction ( Eaton and
Brewer, 1974 ). The selective oxidation of a renewable SH
group helps limit irreversible damage to RBCs that would
otherwise occur. GSSG accounts for only about 0.2% of the
total glutathione in normal human RBCs ( Beutler, 1984 ).
GSH is easily oxidized during the process of sample preparation
and handling for GSSG assays; therefore, care must
be taken not to produce artifactual increases in GSSG concentration
( Rossi et al. , 2002 ). RBC membranes are not
permeable to GSH, but GSSG is exported from RBCs using
an ATP-dependent multidrug resistance protein (MRP)
transporter ( Keppler et al. , 1998 ). The half-life of glutathione
in dog and rabbit RBCs (2 to 5 days) is similar to
that in human RBCs (4 days), whereas longer times (10
to 12 days) are reported in sheep RBCs. The GSSG transport
rate may be the main determinant of glutathione turnover
in RBCs ( Smith, 1974 ). Intracellular GSSG that is not
exported from RBCs or reduced to GSH reacts with protein
SH (PSH) groups to form glutathione-protein mixed disulfides
(GS-SP) according to the reaction GSSG PSH →
GS-SP GSH (Di et al. , 1998 ).
3 . Glutathione Reductase
GSSG produced by the various oxidative reactions is
reduced to GSH by NADPH and the flavin adenine dinucleotide
(FAD)-dependent glutathione reductase (GR) reaction.
Riboflavin metabolism affects the availability of FAD
and the fraction of the protein that exhibits activity ( Beutler,
1989 ; Harvey and Kaneko, 1975b ). GR activity is unmeasurable
in RBCs from horses that are deficient in FAD secondary
to a defect in RBC riboflavin metabolism ( Harvey
et al. , 2003 ). NADPH is produced by the initial enzyme
reactions of the pentose phosphate pathway, and RBCs
increase pentose phosphate pathway metabolism in response
to oxidants to provide the NADPH necessary for the regeneration
of GSH. Excepting the cat, RBCs of domestic animals
have lower GR activity than those of humans ( Agar et al. ,
1974b ; Harvey and Kaneko, 1975b ). RBCs from horses are
slower than RBCs from other species studied in their ability
to regenerate GSH after it has been oxidized in vitro . Horses
also appear less able to protect their RBCs against oxidative
injury induced by incubation with high levels of ascorbate,
which stimulates the GR reaction by the oxidation of GSH.
These reduced abilities may be related to the finding that
horses have lower RBC GR activities than RBCs from
humans and most domestic animal species. In addition, the
K m of GSSG for GR is higher in horses than in three other
species measured ( Harvey and Kaneko, 1975b ).
4 . Glutathione S-Transferase
Glutathione S-transferase (GST) catalyzes the formation
of glutathione S-conjugates between GSH and certain
electrophilic substrates. The same ATP-dependent MRP
transport system appears to transport GSSG and glutathione
S-conjugates out of RBCs ( Keppler et al. , 1998 ). GST
is susceptible to inactivation by electrophilic compounds;
consequently, RBC GST activity may provide a marker for
certain types of chemical exposure ( Ansari et al. , 1987 ).
GST activity is present in RBCs of all mammalian species
studied thus far, but natural electrophilic substrates and the
potential involvement of these glutathione S-conjugates in
glutathione turnover in normal animals are unknown ( Board
and Agar, 1983 ; Vodela and Dalvi, 1997 ). A direct correlation
between GST activity and GSH concentration has been
reported in sheep and dog RBCs ( Goto et al. , 1992 ). This is
probably related to the fact that GST is stabilized by GSH.
The GST-mediated conjugation of various carcinogens
and other electrophilic drugs in the liver is important in
the protection of the body against these agents ( Chasseaud,
1979 ), but the importance of this activity in protecting RBCs
against xenobiotics remains to be documented. The distribution
of GST isozymes varies in tissues, including RBCs. As
a result, RBCs from different individuals (even within the
same species) may vary in their conjugating abilities with
various xenobiotics ( Ploemen et al. , 1995 ). GST can also
bind free hemin that is released during Hb oxidation, presumably
reducing damage to RBC membranes ( Harvey and
Beutler, 1982 ).
5 . Glutathione Peroxidase
Low levels of H 2 O 2 are produced in the course of normal
cellular events and higher levels may be generated
by exogenously administered redox active compounds
( Saltman, 1989 ). GPx catalyzes the conversion of H 2 O 2 to
H 2 O ( Fig. 7-5 ). It also catalyzes the reduction of fatty acid
hydroperoxides, and 1-monoacylglycerol hydroperoxides
( Thomas et al. , 1990 ). Another GPx in RBCs termed phospholipid
hydroperoxide glutathione peroxidase participates
in the reduction of more complex phospholipid hydroperoxides
using GSH ( Fujii et al. , 1984 ).
Selenium is incorporated as selenocysteine at the active
site of a wide range of selenoproteins, including GPx,
phospholipid hydroperoxide glutathione peroxidase, and
thioredoxin reductase in RBCs ( Brown and Arthur, 2001 ).
GPx activity in RBCs correlates directly with blood selenium
concentration in ruminants, horses, and rats but not
in pigs or higher primates ( Anderson et al. , 1978 ; Beilstein