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Chapter 30

Erythrocytic Protection from O2 Toxicity

Overview
Breathing 100% oxygen can be toxic.
Superoxide anions (O ., i.e., free radicals) are produced normally in the body, and
2

are usually quickly removed through several different cellular processes.

Hydroxyl radicals (OH.) are considered to be more toxic than superoxide anions or
hydrogen peroxide (H2O2).
Hydroperoxidation of membrane lipids adversely affects membrane structure
and function.
Catalase, superoxide dismutase (SOD), glutathione peroxidase, and methemoglobin
reductase protect erythrocytes from free radicals.
Excessive oxidative stress may result in the formation of Heinz bodies in
circulating erythrocytes.
Vitamin E, vitamin C, and perhaps uric acid also act as antioxidants.
Erythrocytes, or red blood cells (RBCs), carry
oxygen (O2) from the lungs to tissues, and are
involved in the transport of carbon dioxide
(CO2) from tissues to the lungs (see Chapter 84).
The mature erythrocyte is a highly specialized
cell. Hemoglobin (Hb), which constitutes about
one-third of its weight, contributes red color and
gas-carrying capacity (see Chapter 31). Although
RBCs of primates may have a 120-day life-span
(traveling some 175 miles through the circulation), the life-span of RBCs among domestic
animal species varies considerably (e.g., 65
days in the pig and 150 days in the horse). In
smaller animals with a high metabolic rate, the
survival time is generally shorter than in larger
animals, and red cell survival time generally
increases with hibernation. On average, approximately 1% of circulating erythrocytes are
replaced daily.
Copyright 2015 Elsevier Inc. All rights reserved.

As erythrocytes age, they are removed from

the circulation by cells of the reticuloendothelial (RE) system (predominantly macrophages
in the spleen). To balance this degradation,
there is a constant need for hematopoiesis
(production of new RBCs in bone marrow). The
primary stimulatory factor for red blood cell
production is erythropoietin (EPO), a protein
hormone produced largely by interstitial cells
of the inner renal cortex.
Erythrocytes carry on their surface antigenic
determinants commonly known as blood groups.
These determinants are commonly, but not
always, identified with specific carbohydrate
elements of glycoproteins on the cell surface.

Oxygen Toxicity

Although O2 is essential for mammalian life,

breathing 100% oxygen for several hours can

34 Chapter 30

be toxic, with damage to the lungs, brain, and

retina occurring. At one time this was a major
cause of blindness among premature human
infants with respiratory distress syndrome.
Now, such infants are administered air
containing no more than 40% oxygen.
The toxicity of O2 is due to the production of
highly reactive products derived from it. Even
under normal circumstances, small amounts
of these reduced products are produced;
however, RBCs and other tissues are specialized for their removal. These reactive products
are the superoxide anion (O2., a free radical),
hydrogen peroxide (H2O2), and the hydroxyl
radical (OH., Fig. 30-1). The O2. normally has
a short half-life (milliseconds), and arises from
the acquisition of a single electron (e-) by
a molecule of O2. It can be generated by
the action of ionizing radiation on O2 (thus
causing tissue radiation damage), from
xanthine oxidase (an enzyme which catalyzes
the oxidation of xanthine to uric acid, see
Chapter 17), from flavoprotein oxidases (e.g.,
aldehyde oxidase), from auto-oxidation of
reduced quinones, catecholamines and thiols
(i.e., oxidations in the absence of an enzyme),
and when O2 combines with Hb or myoglobin.
In contrast to the recent appreciation of the
role of superoxide anions, generation of H2O2
in cellular oxidations (and the need to reduce
the concentration of this toxic metabolite) has
been known for many years. Enzymes known
to generate H2O2 include D-amino acid oxidase
and amine oxidase, as well as superoxide
dismutase (SOD, see below).

The hydroxyl radical (OH.) can be generated

in erythrocytes from H2O2 in both the HaberWeiss and Fenton reactions (see Fig. 30-1). It
is considerably more reactive, and therefore
more toxic than either H2O2 or O2.. It affects
proteins and DNA, as well as unsaturated fatty
acids in cell membrane phospholipids (Fig.
30-2 and Chapter 46). Hydroperoxidation of
membrane lipids has two consequences:
1) It increases the hydrophilic nature of the
lipid, which changes membrane structure
so that normal function is disturbed.
2) It inhibits some enzymes, thus compromising
metabolic processes within the membrane
and within the cell.
For example, when red blood cell membranes
are sufficiently damaged by hydroperoxidation,
cells are more rapidly degraded with resultant
anemia. A further problem is that iron atoms in
Hb are readily oxidized (even by H2O2), and the
resulting methemoglobin is unable to properly
transport O2.

Cellular Protection Against Free

Radicals

At least four cellular mechanisms appear to

play a role in reducing harmful effects of these
oxidants (Fig. 30-3). Indeed, at normal oxygen
tensions they usually eliminate the problem
entirely.
The enzyme SOD serves to lower normal
intracellular O2. concentrations to extremely
low levels (<10-11 M). The H2O2 produced is
then removed by the action of catalase, or

Figure 30-1

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Erythrocytic Protection from O2 Toxicity

Figure 30-2

Figure 30-3
glutathione peroxidase. SOD, a zinc- and
copper-containing enzyme, is present in all
aerobic tissues, as well as RBCs (see Chapters
49 and 50).
Catalase is a rather ubiquitous enzyme, that
selectively converts H2O2 to O2 and H2O. Patients
deficient in this enzyme, however, show few
toxic symptoms, presumably because of the
redundant actions of glutathione peroxi-

dase.
The tripeptide glutathione is present at high
concentrations in RBCs (see Chapter 31). The
reduced form of glutathione is represented
by the abbreviation GSH, thus highlighting
the importance of the sulfhydryl (-SH) group
contributed by cysteine. This group is highly
reactive, and may also act non-enzymatically as a free radical scavenger (as well as

34 Chapter 29

The liver is a major producer of reduced glutathione (GSH) for its own use, and
for that of other tissues. Following production, GSH is released into both blood
and bile. Note: RBCs are capable of producing their own GSH.

Methionine
Liver
Cystathionine
Gut
Cysteine
ATP

Glutamate

ATP

Glycine

Glutathione (GSH)

GSHbile
Bile duct
GSHlumen

GSHblood

Enterocytes

distributed to
other tissues

Figure 30-4
protecting against H 2O 2 via conversion to
H2O). This vitally important enzyme contains
selenium (Se), an essential mineral in the
diet (see Chapter 51). In normal RBCs, GSH is
constantly being oxidized to GSSG, but GSSG
accounts for less than 1% of total erythrocyte
glutathione. Continual reduction of GSSG to
GSH is accomplished by the FAD-containing
enzyme, glutathione reductase, and NADPH
(produced in the HMS); reduced glutathione
(GSH) again becomes available to protect
against oxidizing agents. The liver also
produces GSH, and exports it into blood and
bile for use by other tissues (Fig. 30-4).
Methemoglobin reductase does not
use NADPH, instead it makes use of NADH
generated during anaerobic glycolysis in the
RBC (see Chapter 31). Methemoglobin (metHbFe+++) is an inactive form of Hb in which the iron
has been auto-oxidized from the ferrous (Fe++)

to ferric (Fe+++) state by superoxides. Methemoglobin is dark-colored, and when present

in large quantities in the circulation causes
a dusky discoloration of the skin resembling
cyanosis. Only heme iron in the ferrous state
can carry oxygen reversibly (HbFe++ + O2 <>
HbFe++-O2), but small amounts of Hb-Fe++ still
undergo slow auto-oxidation (even in the
absence of oxidative stress) to metHb-Fe+++.
Methemoglobin reductase converts it back to
its functional form. Methemoglobinemia due to
methemoglobin reductase deficiency may be
inherited or acquired (by ingestion of certain
drugs and/or other chemicals).
Excessive oxidative stress may result in the
formation of Heinz bodies (large rigid structures that distort the RBC membrane). Heinz
bodies are formed when the SH groups of Hb
become oxidized, and the globin precipitates.
Horses that feed on red maple leaves, for
example, sometimes develop an acute hemolytic

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Erythrocytic Protection from O2 Toxicity

anemia that is characterized by the formation

of Heinz bodies and methemoglobin in erythrocytes. The oxidizing agent in red maple leaves
has not been clearly elucidated.
Heinz body formation will generally occur
within 24 hours of toxin exposure, and may
occur with or without methemoglobinemia.
Oxidative injury to erythrocytic membranes
may also occur, with changes usually being
irreversible, thus altering membrane deformability. The spleen generally functions to remove
Heinz bodies from erythrocytes; consequently,
if the spleen is removed, Heinz bodies persist
in the circulation for longer periods of time.
Compared to other domestic animals, the
spleen of the cat is generally less capable
of removing Heinz bodies from erythrocytes.
Therefore, these inclusions may be seen in the
blood of normal cats. Acetaminophen toxicity is
a common cause of Heinz body anemia in cats.
Because acetaminophen is normally detoxified
in the liver via glucuronidation, and cats are
less capable of glucuronidating xenobiotics,
the circulating half-life of this drug increases,
thus exacerbating the toxicity (see Chapter 29).
Onion ingestion is known to precipitate Heinz
body formation in cattle, horses, dogs, and
cats. The toxic agent is reported to be n-propyl
disulfide, a compound which decreases erythrocytic glucose-6-phosphate dehydrogenase
activity, thus decreasing NADPH generation
and GSH production.
Vitamin antioxidants available to the body
include tocopherol (vitamin E, see Chapter
46), and ascorbate (vitamin C, see Chapter
39). Several problems associated with red
cell glucose 6-phosphate dehydrogenase
deficiency (see Chapter 31) have reportedly
improved following vitamin E administration.
Uric acid is also reported to possess antioxidant activity, and may contribute to longevity in
certain vertebrate species.

OBJECTIVES
Describe the primary functions of erythrocytes,
and discuss their turnover rate.
Show how superoxide anions, H2O2 and hydroxyl
radicals are normally generated in the body.
Identify the reactants and products of the
Haber-Weiss and Fenton reactions.
Explain why hydroperoxidation of red cell membranes can lead to anemia.
Know the sequential roles played by SOD, catalase (C) and glutathione peroxidase (GP) in cellular protection against free radicals.
Understand why selenium is important to the
health of animals.
Recognize how methemoglobin reductase helps
to increase oxy-hemoglobin binding, and why it
is needed for NAD+ generation in erythrocytes.
Explain how Heinz bodies can be formed in animals.
Indicate how vitamin E or vitamin C ingestion
might improve the condition of an animal with
Glc-6-PD deficiency.
Explain how enterocytes of the digestive tract
receive reduced glutathione.
Know how and why methemoglobinemia develops.
Recognize why catalase and glutathione peroxidase are somewhat redundant enzymes.
Recognize the association of reperfusion injury
with free radical accumulation (see Case Study
#3).
QUESTIONS
1. Which one of the following reactive products
derived from oxygen is considered to be the
most toxic?
a. The superoxide anion (O2.)
b. Hydrogen peroxide (H2O2)
c. The hydroxyl radical (OH.)
d. Water (H2O)
e. Superoxide dismutase (SOD)

34 Chapter 30

6. b
5. c
4. d
3. e
2. d
1. c
ANSWERS

12. The half-life of the superoxide anion

is normally:
a. Milliseconds.
b. Seconds.
c. Minutes.
d. Hours.
e. Days

7. a

7. H2O2 is normally detoxified by:

a. Catalase and glutathione peroxidase.
b. SOD and glutathione reductase.

11. Methemoglobin reductase is needed to:

a. Continually return the oxidized form of
iron in hemoglobin to the
ferrous state.
b. Convert erythrocytic hydrogen
peroxide to water.
c. Peroxidize unsaturated fatty acids
in red cell membranes.
d. Continually auto-oxidize the iron
in erythrocytic hemoglobin.
e. Form GSH and NADP+ from
GSSG, H+ and NADPH in
erythrocytes.

8. e

6. Methemoglobin is:
a. The active form of hemoglobin in which
the iron has been auto-oxidized from the
ferric (Fe+++) to ferrous (Fe++) state.
b. Dark-colored (compared with hemoglobin).
c. Not normally formed in erythrocytes.
d. Formed by the action of methemoglobin
reductase on oxygenated hemoglobin.
e. Normally found in blood plasma, but not
in red blood cells.

10. Heinz bodies are:

a. Free radicals.
b. Precipitated hemoglobin molecules.
c. Oxidized glutathione molecules.
d. Methemoglobin molecules.
e. None of the above

9. c

5. Which one of the following erythrocytic

enzymes uses NADH?
a. Catalase
b. Phosphofructokinase
c. Methemoglobin reductase
d. Glutathione peroxidase
e. Superoxide dismutase

9. Reperfusion injury following myocardial

ischemic injury is associated with increased
levels of which of the following:
a. Superoxide dismutase.
b. Catalase.
c. Superoxide and hydroxyl radicals.
d. Glutathione peroxidase.
e. All of the above

10. b

4. Which one of the following is a selenium

containing enzyme in erythrocytes?
a. Hexokinase
b. Phosphofructokinase
c. Methemoglobin reductase
d. Glutathione peroxidase
e. Superoxide dismutase

8. The usual CBC of a patient on glucocorticoid

therapy would be expected to show a:
a. Neutrophilia and lymphopenia.
b. Erythrocytosis.
c. Hyperglycemia.
d. Thrombocytosis.
e. All of the above

11. a

3. Which one of the following is an erythrocytic

zinc- and copper-containing enzyme?
a. Hexokinase
b. Catalase
c. Methemoglobin reductase
d. Glutathione peroxidase
e. Superoxide dismutase

c. Methemoglobin reductase and catalase.

d. The Haber-Weiss and Fenton reactions.
e. Glutathione peroxidase and SOD.

12. a

2. Hydroperoxidation of membrane lipids has

which one of the following consequences for
a cell?
a. It increases the lipophilic nature of the
lipid, thus increasing transport of fat-soluble compounds into and out of the cell.
b. It increases enzyme activity within the cell
membrane.
c. It causes fatty acids contained in
membrane-bound phospholipids to become
more saturated.
d. It increases the hydrophilic nature of the
lipids, thus changing membrane structure
and decreasing function.
e. It decreases free radical formation within
the membrane.

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