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

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Chapter | 7 The Erythrocyte: Physiology, Metabolism, and Biochemical Disorders
Methylene blue (MB) is used to treat toxic methemoglobinemia
because it causes MetHb to be reduced faster than
occurs by the relatively slow Cb 5 R reaction. MB is reduced
to leukomethylene blue (LMB) by the NADPH-dependent
diaphorase discussed previously ( Fig. 7-5 ), and LMB reacts
spontaneously with MetHb, reducing it to Hb and regenerating
MB ( Sass et al. , 1969 ). MB was suggested to be of limited
value in the treatment of methemoglobinemia in horses,
as it had a limited ability to stimulate MetHb reduction in
horse RBCs in vitro using glucose as the substrate ( Robin
and Harley, 1966 ). However, MB was subsequently found
to be effective in reducing methemoglobinemia in horses in
vivo ( Dixon and McPherson, 1977 ; Harvey et al. , 2003 ).
Being a redox dye, MB can participate in various oxidative
reactions on entering RBCs. It is important that it
be used judiciously lest it potentiate HzB body formation
and hemolysis that might result from the original oxidative
insult ( Harvey and Keitt, 1983 ).
V . DETERMINANTS OF RBC SURVIVAL
A wide variety of conditions, including immune-mediated
hemolytic anemias and infectious diseases, result in shortened
RBC survival. Readers are referred to Schalm’s
Veterinary Hematology (Feldman et al. , 2000 ) for information
concerning conditions beyond the scope of this chapter.
A . Oxidative Injury
Oxidants produce different patterns of intracellular and
membrane damage. For example, nitrite produces MetHb
with minimal damage to Hb or membranes ( May et al. ,
2000 ); onion toxicity produces Heinz bodies and membrane
damage with minimal methemoglobinemia in dogs
( Harvey and Rackear, 1985 ); and acetaminophen toxicity
results in significant methemoglobinemia, Heinz body formation,
and membrane damage in cats ( Finco et al. , 1975 ).
Different patterns may be related to differences in lipid solubility,
redox potentials, reactivity with SH groups, binding
to heme, and the source or site of oxidant generation.
Extracellularly produced oxidants can damage the membrane
before reaching the cytosolic protective mechanisms.
Oxidants that are generated intracellularly in coupled reactions
with OxyHb tend to produce more Hb injury than
membrane injury. Hb has been described as a “ frustrated ”
oxidase, because it potentiates the generation of oxidants
by a variety of drugs ( Carrell et al. , 1977 ).
1 . MetHb Formation
Although MetHb formation is reversible and does not
enhance RBC destruction per se, it is a component of oxidative
injury to RBCs and is generally involved as a step in
HzB formation. Clinical signs associated with methemoglobinemia
are the result of hypoxia because MetHb cannot
bind O 2 . Lethargy, ataxia, and semistupor do not become
apparent until MetHb content exceeds 50%, with a comalike
state and death ensuing when it exceeds 80% ( Bodansky,
1951 ). The cyanotic appearance of mucous membranes
associated with this condition may not be easily recognized
in heavily pigmented animals. MetHb content is quantified
spectrophotometrically, but a spot test can determine
whether clinically significant levels of MetHb are present.
One drop of blood from the patient is placed on a piece of
absorbent white paper and a drop of normal control blood is
placed next to it. If the MetHb content is 10% or greater the
patient’s blood should have a noticeably brown coloration.
Approximately 3% of Hb is spontaneously oxidized to
MetHb each day in normal people ( Mansouri and Lurie,
1993 ) and dogs ( Harvey et al. , 1991 ). A similar rate of
MetHb formation (4%) has been calculated for mice ( Johnson
et al. , 2005 ). This MetHb formation results primarily from
spontaneous autoxidation of OxyHb ( Johnson et al. , 2005 ).
Although the iron moiety of DeoxyHb is in the ferrous
state, in OxyHb it exists in (or near) the ferric state, with
an electron being transferred to the O 2 molecule to give a
bound superoxide (·O 2
) ion ( Mansouri and Lurie, 1993 ).
During deoxygenation, the electron returns to the iron moiety
and O 2 is released. Autoxidation to MetHb with the
release of ·O 2
occurs when the bound ·O 2
is replaced by a
nucleophile such as C1 (Wallace et al. , 1974 ).
Oxidants such as hexavalent chromates, chlorates,
cobalt, and copper oxidize Hb iron by extracting electrons
( Umbreit, 2007 ). Cupric ions oxidize the reactive SH group
on the beta chains of human and animal Hbs ( Taketa and
Antholine, 1982 ), and then an electron is transferred from
heme iron to regenerate the SH group. Cupric ion is regenerated
by interaction with oxygen. The series of reactions
by which copper catalyzes Hb oxidation to MetHb ( Carrell
et al. , 1978 ) are as follows:
Cu2 .
Fe2S → Cu1Fe2S
.
Fe2S → Fe3S
Cu1
O → Cu2
. O
2
These reactions occur rapidly but continue only until about
50% of total hemes are oxidized because alpha chains
are resistant to oxidation ( Taketa and Antholine, 1982 ).
Additional oxidative reactions involving copper can denature
Hb and damage membranes ( Hochstein et al. , 1978 ).
Rather than extracting electrons, many oxidant drugs
produce MetHb by donating electrons to OxyHb ( Carrell
et al. , 1977 ), as is shown below for phenylhydroxylamine
(PHA):
Hb
Hb
. O PHA Hb O PHA
.
2 →
2 2
. O 2H → Hb H O
3. 3. .
3.
2 2 3
2 2
Drug free radicals, such as PHA, that are generated can
donate the unpaired electron to molecular oxygen to form
2