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

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Chapter | 7 The Erythrocyte: Physiology, Metabolism, and Biochemical Disorders
2002 ; Lykkesfeldt, 2002 ), and ischemia/reperfusion ( Valko
et al. , 2007 ).
1 . Reactive Oxygen Species
From a thermodynamic standpoint, oxygen is a strong oxidant,
but its reactivity is limited by virtue of its unusual electronic
configuration ( Green and Hill, 1984 ). Although it is
relatively unreactive, oxygen can be metabolized in vivo to
form highly reactive derivatives. A single-electron reduction
of O 2 yields the superoxide ( . O 2
) free radical. The
major source within RBCs appears to be the autoxidation of
OxyHb ( Johnson et al. , 2005 ). There are a wide variety of
other sources of ·O 2
production in various tissues; examples
include the ubiquinone-cytochrome b region in mitochondria;
uncoupled cytochrome P -450 reactions; autoxidation
of adrenaline, certain flavins, and SH groups; enzyme
reactions such as xanthine oxidase and tryptophan deoxygenase;
and the oxidant burst of activated neutrophils and
mononuclear phagocytes ( Freeman and Crapo, 1982 ). In the
absence of inflammation, the major source of . O 2
production
in nonerythroid cells is the mitochondrion ( Johnson
et al. , 2005 ). Superoxide undergoes spontaneous and enzyme
catalyzed dismutation to H 2 O 2 . In the presence of a metal
ion such as iron or copper, . O 2
and H 2 O 2 interact to form
the highly reactive hydroxyl radical ( . OH). Additional ROS
include singlet O 2 (an excited state of O 2 ), ozone (O 3 ) and
hypochlorous acid (HOCl), generated by myeloperoxidase
in neutrophils from H 2 O 2 , and Cl (Pryor et al. , 2006 ).
2 . Reactive Nitrogen Species
Nitric oxide is a stable free radical generated from arginine
by the nitric oxide synthase (NOS) reaction. Three isoforms
of NOS exist. The endothelial enzyme (eNOS) and the neuronal
enzyme (nNOS) are constitutively expressed. The third
form (iNOS) is induced in a variety of cells, often as a result
of immunologic/inflammatory stimulation ( Pryor et al. ,
2006 ). Nitric oxide is a second messenger involved in a
variety of biological functions. Within the vasculature, it
promotes vasodilation and inhibits platelet aggregation and
leukocyte adhesion to the endothelium. It also functions as
a neurotransmitter and has antimicrobial functions in phagocytes.
The half-life of . NO in the circulation is believed to
be 0.1sec ( Rifkind et al. , 2006 ). It combines with oxygen
to produce additional oxidants, nitrogen dioxide ( . NO 2 ) and
dinitrogen trioxide (N 2 O 3 ), which are eventually converted
to nitrite (NO 2
). Nitric oxide reacts with . O 2
, to form the
strong oxidant peroxynitrite (ONOO ). Peroxynitrite combines
with CO 2 to form ONOOCO 2
, which decomposes to
form . NO 2 and the carbonate radical ( . CO 3 ).
Nitric oxide exhibits complex interactions with Hb to form
several products, including nitrosylhemoglobin [Hb(II)NO],
S-nitrosylated Hb, MetHb, and nitrate, depending on
whether it reacts with OxyHb or DeoxyHb ( Rifkind et al. ,
2006 ). The RBC was considered to be an important . NO
scavenger in earlier literature, but much of the . NO apparently
does not enter the cytoplasm of RBCs, primarily because
their membranes and associated cytoskeleton layers limit
its uptake ( Han et al. , 2005 ). However, free Hb in plasma
scavenges . NO up to 600 times more readily than Hb within
RBCs. Consequently, intravascular hemolysis disrupts . NO
homeostasis, which may lead to vasoconstriction, decreased
blood flow, platelet activation, increased endothelin-1
expression, and organ injury ( Gladwin et al. , 2004 ).
3 . Drugs, Environmental Agents, and
Metabolic Intermediates
A wide variety of drugs, environmental agents, and metabolic
intermediates either exist as free radicals or can be
converted to free radicals by cellular metabolic processes
( Freeman and Crapo, 1982 ; Mason, 1982 ). These free radicals
can be more damaging than the ROS and RNS species
listed previously.
L . Metabolic Protection against Oxidants
1 . Superoxide Dismutase
Superoxide dismutase (SOD) is a copper- and zinccontaining
enzyme that was first isolated from cattle blood
( McCord and Fridovich, 1969 ). It promotes the dismutation
of two . O 2
molecules to H 2 O 2 and O 2 ( Fig. 7-5 ).
SOD helps prevent the buildup of superoxide, which can
act as an oxidant by itself, or combine with H 2 O 2 to form
the . OH radical, or combine with . NO to form peroxynitrite
( Pryor et al. , 2006 ). The activity of SOD in RBCs of
domestic animals is about the same as or higher than that
in humans ( Harvey and Kaneko, 1977 ; Suzuki et al. , 1984 ).
RBC SOD activity is reduced in animals fed diets deficient
in copper ( Andrewartha and Caple, 1980 ; Williams et al. ,
1975 ). Zinc is also needed for optimal activity, and consequently,
SOD activity may also be low in zinc-deficient
animals ( Hirose et al. , 1992 ).
2 . Glutathione
Reduced glutathione (GSH) is of central importance in the
protection against oxidant injury. It is a tripeptide of glutamic
acid, cysteine, and glycine that occurs in approximately
2 mM concentrations in RBCs. It is synthesized
de novo in RBCs of humans and animals from constituent
amino acids via two ATP-requiring reactions, utilizing
gamma-glutamylcysteine synthetase and glutathione synthetase
( Beutler, 1989 ). GSH has a highly reactive (easily oxidizable)
sulfhydryl (SH) group that, like other thiols, may
act nonenzymatically as a free radical acceptor to counteract
oxidant damage ( Prins and Loos, 1969 ). GSH can
also bind free hemin (containing iron as Fe 3 ) that may be