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

IV. Mature RBC
207
reduced by ascorbate, presumably at the aqueous-lipid interface
of the lipid bilayer ( May, 1998 ). Vitamin E deficiency
increases the susceptibility of RBCs to peroxidative hemolysis
( Duthie et al. , 1989 ; Pillai et al. , 1992 ). Its location in the
membrane provides little protection against cytosolic oxidative
injury ( Rotruck et al. , 1972 ). Vitamin E also inhibits
hemolysis induced by hemin, purportedly by functioning as
a membrane-stabilizing agent ( Wang et al. , 2006 ).
9 . Vitamin C (Ascorbate)
Vitamin C is an antioxidant with high reducing potential. It
donates one or two electrons to a variety of oxidants, including
oxygen free radicals and peroxides. It also appears
to have an important function in the reduction of oxidized
vitamin E within RBC membranes ( May, 1998 ). The single
electron-oxidized form is a stable free radical that can either
donate or accept electrons. Dehydroascorbate, the two electron-oxidized
form, can be reduced back to ascorbate nonenzymatically
by GSH and enzymatically via the glutaredoxin
reaction and by a separate GSH-dependent dehydroascorbate
reductase enzyme ( Xu et al. , 1996 ). Dehydroascorbate
can also be reduced by the NADPH-dependent thioredoxin
reductase reaction ( May, 1998 ). Ascorbate enters and leaves
cells slowly, but dehydroascorbate is rapidly taken up by
RBCs by facilitated diffusion on the glucose transport protein
(GLUT-1) and reduced to ascorbate within RBCs.
Ascorbate is a major antioxidant in plasma. Ascorbate within
human RBCs readily donates electrons to extracellular
ascorbate free radicals via a plasma membrane redox system
(possibly involving a membrane cytochrome b561), which
helps prevent depletion of extracellular ascorbate. However,
this membrane redox system appears to be less active in
mouse RBCs ( Su et al. , 2006 ).
10 . Lipoic Acid
Lipoic acid is a sulfur-containing compound that is absorbed
from the diet and synthesized in mitochondria ( Smith
et al. , 2004 ). Upon entering cells, it can be reduced to the
dithiol form dihydrolipoic acid by the thioredoxin system
( Smith et al. , 2004 ). Reduction of lipoic acid in human
RBCs is reported to occur via GSH and glutathione reductase
( Constantinescu et al. , 1995 ). Dihydrolipoic acid scavenges
various ROS and RNS and chelates heavy metals;
however, it is present in much lower concentrations in tissues
compared to GSH and ascorbate. Consequently, the
importance of its function as an antioxidant in vivo is questionable
( Smith et al. , 2004 ).
11 . MetHb Reduction
MetHb differs from Hb only in that the iron moiety of the
heme groups is in the ferric rather than the ferrous state.
MetHb forms in vivo at low levels normally and at much
FIGURE 7-9 Methemoglobin reduction pathway. Abbreviations: G3P,
glyceraldehyde-3-phosphate; GAPD, glyceraldehyde-3-phosphate dehydrogenase;
PO 4 , inorganic phosphate; 1,3DPG, 1,3-diphosphoglycerate; NAD,
nicotinamide adenine dinucleotide; NADH, reduced nicotinamide adenine
dinucleotide; Cb 5 R, cytochrome b 5 reductase; FAD, flavin adenine
dinucleotide; Cb 5 -Fe
3
, ferricytochrome b 5 ; Cb 5 -Fe 2 , ferrocytochrome
b 5 ; Hb-Fe
3
, methemoglobin; and Hb-Fe 2 , deoxyhemoglobin. From
Harvey, 2006 , with permission.
higher levels in the presence of oxidative compounds
( Bodansky, 1951 ).
MetHb is unable to bind oxygen and must be reduced to
Hb to be functional. MetHb is primarily reduced by cytochrome-b
5 reductase (Cb 5 R), also called MetHb reductase.
In this reaction, electrons are transferred from NADH to ferricytochrome-b
5 using FAD as the enzyme-bound prosthetic
group; then the resulting ferrocytochrome-b 5 reduces MetHb
nonenzymatically to Hb ( Higasa et al. , 1998 ) (Fig. 7-9 ).
MetHb reduction is more corrective than protective.
RBCs contain another enzyme, NADPH diaphorase
(NADPH MetHb reductase, NADPH dehydrogenase) that is
capable of MetHb reduction when appropriate electron carriers
are present. In addition to redox dyes, such as methylene
blue, various flavins may function as substrates for
reduction by NADPH, prompting investigators to classify
the enzyme as an NADPH flavin reductase ( Yubisui et al. ,
1980 ). The contribution of this enzyme to MetHb reduction
in human RBCs is believed to be insignificant because flavin
concentrations are normally low ( Hultquist et al. , 1993 ).
The finding that this protein also binds heme, porphyrins,
and fatty acids raises additional questions about its possibly
physiological role in RBCs ( Xu et al. , 1992 ).
Following the oxidation of Hb to MetHb with nitrite
in vitro , horse RBCs reduce MetHb at a slower rate than
those of other domestic animals (except pigs) when glucose
is added as the substrate for energy ( Robin and Harley,
1966 ). RBCs from adult pigs cannot reduce MetHb with
glucose as the substrate because they lack a membrane
glucose transporter ( Kwong et al. , 1986 ). In contrast to
NADP, only about half of the total NAD is normally present
in the reduced (NADH) form ( Zerez et al. , 1987 ). Horse
(Medeiros et al. , 1984 ; Robin and Harley, 1967 ) and pig
( Rivkin and Simon, 1965 ) RBCs utilize lactate better than
glucose to generate NADH (by the LDH reaction) for the
reduction of MetHb. Because lactate occurs in blood and
easily diffuses into RBCs, it may be an important substrate
of MetHb reduction in vivo .