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

IV. Mature RBC
203
mechanisms would result in ·NO production, vasodilation,
and increased blood flow at sites where SO 2 in RBCs is
low ( Crawford et al. , 2006 ).
RBCs also promote vasodilation by the release of micromolar
amounts of ATP, an endothelium-dependent vasodilator,
when SO 2 is low. As oxygen is released from OxyHb,
the increased concentration of DeoxyHb binds to the cytoplasmic
domain of band 3, displacing PFK and other glycolytic
enzymes. It is hypothesized that this results in a
stimulation of glycolysis (see Section IV.G), which results
in increased production and release of ATP ( Jagger et al. ,
2001 ). The precise mechanism of ATP release is unknown,
but band 3 and nucleoside transporter band 4.5 have been
implicated in transport ( Jagger et al. , 2001 ). In addition,
a member of the ATP-binding cassette, the cystic fibrosis
transmembrane conductance regulator (CFTR), appears to
mediate a deformation-induced release of ATP from RBCs
( Gov and Safran, 2005 ; Sprague et al. , 2005 ), and this
transport mechanism may also play a role in SO 2 -mediated
ATP release ( Jagger et al. , 2001 ). Blood levels of ATP
rise and fall within minutes, compared to seconds for . NO
effects. Consequently, . NO and ATP may have complementary
roles in acute local and prolonged systemic hypoxia,
respectively ( Singel and Stamler, 2005 ).
J . Pentose Phosphate Pathway
The pentose phosphate pathway (PPP) generates NADPH,
the major source of reducing equivalents in the protection
of RBCs against oxidative injury. This pathway also
produces ribose 5-phosphate (R5P), which is required for
adenine nucleotide synthesis ( Eaton and Brewer, 1974 ).
The PPP competes with the EMP for the G6P substrate
( Fig. 7-5 ). Normally only about 5% to 13% of glucose
metabolized by RBCs flows through the PPP ( Harvey and
Kaneko, 1976a ), but this flow can be accelerated markedly
by oxidants ( Harvey and Kaneko, 1977 ).
The first step in the metabolism of glucose through the
PPP generates NADPH from the oxidation of G6P in the
glucose-6-phosphate dehydrogenase (G6PD) reaction. An
additional NADPH is generated from the oxidative decarboxylation
of 6-phosphogluconate (6PG) to ribulose 5-
phosphate in the 6-phosphogluconate dehydrogenase (6PGD)
reaction. This is the only known reaction producing CO 2 in
mature RBCs. The remaining reactions in the PPP are nonoxidative
and freely reversible. R5P is produced from ribulose
5-phosphate by the R5P isomerase reaction. The net
effect of the metabolism of 3 molecules of G6P through the
PPP is as follows ( Eaton and Brewer, 1974 ):
3G6P 6NADP
→ 3CO2
2F6P G3P
6NADPH
6H
G6PD is the rate-limiting reaction in the PPP under
physiological conditions. Normally, the G6PD reaction in
intact human RBCs operates at only 0.1% to 0.2% of the
maximal enzyme activity, as determined in hemolysates
under optimal conditions. The low rate of this reaction in
RBCs occurs because of limited substrate availability (especially
NADP ) and because G6PD is strongly inhibited by
NADPH and ATP at physiological concentrations ( Yoshida,
1973 ). The maximal G6PD activities measured in hemolysates
from goat and sheep RBCs are much lower than those
of humans or of other domestic animals ( Tables 7-2 and 7-3 ).
However, this comparatively low enzyme activity does not
render sheep RBCs unduly susceptible to the hemolytic
effects of oxidant drugs ( Maronpot, 1972 ; Smith, 1968 ),
in part because ATP does not inhibit G6PD in this species
( Smith and Anwer, 1971 ).
About 91% of total NADP is in the reduced form in
horse RBCs ( Stockham et al. , 1994 ) and 92% to 99% of
total NADP is NADPH in human RBCs ( Kirkman et al. ,
1986 ; Zerez et al. , 1987 ). NADPH is utilized to reduce oxidized
glutathione to GSH, the substrate for the glutathione
peroxidase reaction, and it is bound to catalase, preventing
and reversing the accumulation of an inactive form of catalase
that is generated when catalase is exposed to H 2 O 2
(Kirkman et al. , 1987 ). In the presence of oxidants, NADPH
is oxidized and the PPP is stimulated because the activities
of G6PD and 6PGD are directly related to the concentration
of NADP and inversely related to that of NADPH ( Yoshida,
1973 ). Glutathione metabolism affects PPP activity via
the glutathione reductase (GR) enzyme, which generates
NADP as a result of the reduction of GSSG with NADPH
(Fig. 7-5 ).
K . Nature of Oxidants in Biology
Reactive oxygen species (ROS) and reactive nitrogen species
(RNS) are produced as products of normal cellular
metabolism. They play dual roles as beneficial and deleterious
species. At low to moderate concentrations, . NO and
superoxide ( . O 2
) free radicals are involved in signal transductions
between cells ( Valko et al. , 2007 ). A free radical
is defined as any species with one or more unpaired electrons.
When generated at higher concentrations in diseased
states, these free radicals (and even more potent oxidative
metabolites they produce) can overwhelm protective systems
within the body, producing cellular injury or destruction
( Valko et al. , 2007 ). The oxidants generated vary in
their overall reactivities, and some are fairly selective for
certain biomolecules (e.g., tyrosine, glutathione, linoleic
acid, and ascorbate) ( Pryor et al. , 2006 ).
Increased amounts of endogenous oxidants are generated
in association with various disorders including inflammation
( Lykkesfeldt, 2002 ; Spickett et al. , 1998 ; Weiss
et al. , 1992a ; Weitzman and Gordon, 1990 ), RBC parasites
( Otsuka et al. , 2001 ; Shiono et al. , 2003 ), neoplasia
( Christopher, 1989 ; Della Rovere et al. , 2000 ), diabetes
( Christopher, 1995 ), intense exercise ( Hargreaves et al. ,