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

V. Determinants of RBC Survival
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superoxide or to another OxyHb molecule to form more
MetHb and H 2 O 2 . To achieve greater stability, free radicals
may also extract electrons by oxidizing SH groups of
Hb, enzymes, membrane proteins, or GSH; by oxidizing
membrane unsaturated fatty acids; by oxidizing NADPH
or NADH; and possibly by extracting an electron from
DeoxyHb to form MetHb.
Nitrite quickly enters RBCs and equilibrates across their
membranes and then continues to enter RBCs as a consequence
of its intracellular removal. This involves the formation
of nitrate and MetHb in the presence of OxyHb and the
formation of . NO and MetHb in the presence of DeoxyHb
( Jensen, 2005 ). The reactions involved in MetHb formation
in the presence of nitrite are complex ( Titov and Petrenko,
2005 ; Umbreit, 2007 ). MetHb formation by nitrite is significantly
higher in oxygenated than deoxygenated pig RBCs
( Jensen, 2005 ). Nitrite produces MetHb within RBCs, but
otherwise does not exert strong oxidant stress on these cells
(May et al. , 2000 ). Methemoglobinemia occurs in ruminants
eating nitrate-accumulating plants, especially when
the plants have been fertilized with nitrogenous compounds.
Nitrate is relatively nontoxic, but it is reduced to nitrite by
ruminal microorganisms ( Burrows, 1980 ).
Methemoglobinemia can occur in animals following the
topical application of benzocaine-containing products to skin
(Harvey et al. , 1979 ; Wilkie and Kirby, 1988 ), or in laryngeal
(Krake et al. , 1985 ) or nasopharyngeal sprays ( Davis et al. ,
1993 ; Lagutchik et al. , 1992 ). Benzocaine appears to have
only a limited ability to produce other forms of RBC injury,
although HzB formation can occur ( Harvey et al. , 1979 ).
Many compounds can produce MetHb ( Ash-Bernal
et al. , 2004 ; Bodansky, 1951 ; Umbreit, 2007 ), and most
of them also produce variable degrees of Hb denaturation
and membrane injury. Acetaminophen and phenazopyridine
toxicity in cats ( Harvey, 1995 ), red maple toxicity in
horses ( Alward et al. , 2006 ), copper toxicity in sheep ( Soli
and Froslie, 1977 ), and skunk musk absorption in dogs
( Zaks et al. , 2005 ) are examples of drugs or chemicals that
produce prominent methemoglobinemia and Heinz body
hemolytic anemia. Following exposure to oxidants, MetHb
forms within minutes, but HzB take hours to form. If the
oxidant is rapidly metabolized, MetHb content will generally
be reduced to values approaching normal within 24 h
( Harvey and Keitt, 1983 ).
Low levels of MetHb may form in response to endogenous
oxidant generation during inflammation ( Morita et al. ,
1996 ; Ohashi et al. , 1998 ; Weiss and Klausner, 1988 ).
Remarkable methemoglobinemia, with evidence of RBC
membrane oxidant damage, has been described in cattle
infected with Theileria sergenti (Shiono et al. , 2003 ).
2 . HzB Formation
HzB are composed of oxidized denatured Hb. They are
often not recognized on routinely stained blood films,
because they either are not stained or stain similarly to
the remaining intact Hb. If they are of sufficient size (1 to
2 μ m), they may appear as pale inclusions within RBCs
or as nipple-like projections from the surface of RBCs.
HzB can be visualized as dark, refractile inclusions in new
methylene blue “ wet ” preparations and as light blue inclusions
with reticulocyte stains ( Harvey, 2001 ).
The following sequence of oxidant-triggered biochemical
events leading to HzB formation is proposed ( Allen and
Jandl, 1961 ; Chiu and Lubin, 1989 ; Hebbel and Eaton, 1989 ;
Low, 1989 ; Mawatari and Murakami, 2004 ): (1) ferrohemes
are oxidized to ferrihemes (MetHb). (2) Reactive SH groups
of MetHb are oxidized (cysteine 93 of the human Hb
β chain) and mixed disulfide bonds form following interaction
with GSSG (also generated in response to oxidative
stress). This glutathionylation of MetHb may be protective,
because these globin SH groups can be regenerated via the
glutaredoxin, and possibly thioredoxin, systems ( Klatt and
Lamas, 2000 ). (3) As further oxidation continues, normally
“ buried ” SH groups of MetHb are oxidized causing additional
glutathionylation reactions and presumably disulfide
bond formation between globin chains. These reactions are
probably not reversible ( Mawatari and Murakami, 2004 ).
(4) Conformational changes in globin chains result in dissociation
of the tetramer to dimers and monomers and hemichrome
formation (hemichromes have both the ferric iron’s
fifth and sixth coordinate positions occupied by a ligand provided
by the globin chain). (5) Hemichromes bind to band 3
and to a lesser degree other membrane components, forming
clusters of copolymers. (6) The precipitation and accumulation
of denatured globin molecules result in HzB formation.
Precipitation is potentiated by the dissociation of the ferriheme
(hemin) moieties from the hemichromes because the
resultant free globin chains are unstable.
Hemolytic anemias associated with HzB formation
in domestic animals have resulted from a variety of compounds.
Dietary causes include consumption of onions by
cattle ( Lincoln et al. , 1992 ), sheep ( Kirk and Bulgin, 1979 ;
Knight et al. , 2000 ; Verhoeff et al. , 1985 ), horses (Pierce
et al. , 1972 ), cats ( Kobayashi, 1981 ), and dogs ( Harvey and
Rackear, 1985 ; Ogawa et al. , 1986 ), consumption of garlic
by dogs and horses ( Lee et al. , 2000 ; Pearson et al. , 2005 ;
Yamato et al. , 2005 ), and consumption of kale and other
Brassica species by ruminants ( Greenhalgh et al. , 1969 ;
Smith, 1980 ; Suttle et al. , 1987 ). Dipropyl and diallyl di-,
tri-, and tetrasulfides and possibly other organosulfur compounds
derived from plants in the Allium genus (onions,
garlic, and chives) cause oxidative damage to RBCs. ROS
are formed during redox recycling of these compounds
or their metabolites in the presence of GSH and OxyHb
(Munday et al. , 2003 ). The role of GSH as a source of
electrons in this redox cycle may explain why dogs with
high RBC GSH concentrations are more susceptible to
onion-induced RBC damage than dogs with normal RBC
GSH concentrations ( Yamoto and Maede, 1992 ). In the