Oxidative Stress and Haemolytic Anaemia In Dogs and Cats: A Comparative Approach

Abstract

Oxidative stress contributes to Haemolytic Anaemia in many species including dogs and cats, as well as in humans. Red cells are exposed to a continual oxidant challenge, both endogenously from within the red cells themselves and also exogenously from other tissues, and from ingested or administered oxidants. When the oxidative challenge exceeds the antioxidant provisions of the red cell, damage occurs in the form of lipid and protein peroxidation, cytoskeletal crosslinking, oxidation of haemoglobin to methemolglobin, and precipitation of denatured sulphhaemoglobin as Heinz bodies. These deleterious sequelae produce fragile red cells with reduced lifespan, and result in poorer oxygen delivery to tissues, intravascular haemolysis, anaemia, haemoglobinuria and jaundice. A number of features increase the risk of oxidant damage in dogs and cats. Thus dog red cells have low levels of the antioxidant enzyme catalase. Cat haemoglobin has at least four times as many readily oxidizable thiol residues compared to most species, whilst their hepatic capacity for glucuronidation is much reduced, which can result in greater accumulation of oxidants. Like humans, both species may also be exposed to excess oxidants from systemic diseases such as diabetes mellitus, hepatic lipidosis, hypophosphatemia and neoplasias. Iatrogenic oxidants include drugs such as acetaminophen and other non-steroidal anti-inflammatory compounds. Ingested toxins include heavy metals, particularly important in dogs with their increased propensity for scavenging. Ingestion of feeds containing products from Allium species of plants has also long been associated with red cell oxidative damage and Heinz body formation in both dogs and cats. Though less common than in humans, there are occasional congenital enzyme deficiencies which reduce the enzymatic oxidant defence of the red cells in these species. Treatment usually relies on removal of the oxidant challenge or support against the resulting anaemia. Specific antioxidants currently lack efficacy but analogy with human medicine suggests that a range possible antioxidants may be potentially beneficial.

Full text

Integrave Journal of Veterinary Biosciences
Volume 3 Issue 3
Integr J Vet Biosci, Volume 3(3): 1–5, 2019
Research Open
Review Article
Oxidative Stress and Haemolytic Anaemia In Dogs and
Cats: A Comparative Approach
Gibson JS1*, Wadud R1, Lu DCY1, Brewin JN2 and Rees DC2
1Department of Veterinary Medicine, Madingley Road, Cambridge, UK
2Department of Paediatric Haematology, King’s College Hospital, London, UK
*Corresponding author: John S. Gibson, Department of Veterinary Medicine, Madingley Road, Cambridge, CB3 0ES, UK; E-mail: jsg1001@cam.ac.uk
Received: October 15, 2019; Accepted: October 23, 2019; Published: October 31, 2019
Abstract
Oxidative stress contributes to Haemolytic Anaemia in many species including dogs and cats, as well as in humans. Red cells are exposed to a continual
oxidant challenge, both endogenously from within the red cells themselves and also exogenously from other tissues, and from ingested or administered
oxidants. When the oxidative challenge exceeds the antioxidant provisions of the red cell, damage occurs in the form of lipid and protein peroxidation,
cytoskeletal crosslinking, oxidation of haemoglobin to methemolglobin, and precipitation of denatured sulphhaemoglobin as Heinz bodies. These
deleterious sequelae produce fragile red cells with reduced lifespan, and result in poorer oxygen delivery to tissues, intravascular haemolysis, anaemia,
haemoglobinuria and jaundice. A number of features increase the risk of oxidant damage in dogs and cats. Thus dog red cells have low levels of the
antioxidant enzyme catalase. Cat haemoglobin has at least four times as many readily oxidizable thiol residues compared to most species, whilst their
hepatic capacity for glucuronidation is much reduced, which can result in greater accumulation of oxidants. Like humans, both species may also be
exposed to excess oxidants from systemic diseases such as diabetes mellitus, hepatic lipidosis, hypophosphatemia and neoplasias. Iatrogenic oxidants
include drugs such as acetaminophen and other non-steroidal anti-inammatory compounds. Ingested toxins include heavy metals, particularly
important in dogs with their increased propensity for scavenging. Ingestion of feeds containing products from Allium species of plants has also long been
associated with red cell oxidative damage and Heinz body formation in both dogs and cats. Though less common than in humans, there are occasional
congenital enzyme deciencies which reduce the enzymatic oxidant defence of the red cells in these species. Treatment usually relies on removal of the
oxidant challenge or support against the resulting anaemia. Specic antioxidants currently lack ecacy but analogy with human medicine suggests that
a range possible antioxidants may be potentially benecial.
Key words: Antioxidant Defence, Dogs and Cats, Haemolytic Anaemia, Oxidative Stress
Introduction
Red cells occupy a unique position within the vertebrate body.
When mature, they are enucleated and lack cytoplasmic organelles
[1]. As such, they are therefore unable to carry out ribosomal protein
synthesis or mitochondrial oxidative phosphorylation. ey are
dependent upon glycolysis (or the Emden-Meyerho pathway) for
whatever ATP supply is required to maintain their osmotic integrity,
through various ion pumps, and for other energy requiring events,
like synthesis of reduced glutathione, one of their main antioxidant
defences [2, 3]. All vertebrate red cells have the main task of carriage
of blood gases, oxygen from respiratory tissues and carbon dioxide
from metabolically active tissues. Notwithstanding, there are some
surprising species dierences in function, which are signicant
both physiologically and pathologically [1]. For example, most
vertebrate red cells contain high levels of K+ and low levels of Na+,
whose gradients are maintained through the functioning of the
ATP-dependent Na+/K+ pump in the red cell membrane. is pump,
together with a normally low passive “leak” to Na+ and K+ prevent
osmotic swelling which would otherwise occur through the large
cytoplasmic load of impermeable protein, especially haemoglobin
(Hb), and other molecules, notably organic phosphates [4]. By
contrast, dog and cat red cells are usually low in K+ and high in Na+.
When mature – but not during development – their red cells lack Na+/
K+ pumping capacity and rather they use combinations of Ca2+ pumps
and Na+/Ca2+ exchange proteins to maintain osmotic equilibrium [5].
An exception is high K+-containing red cells of certain Asian breeds
for example, the Japanese Shibas and Akitas [6] which retain Na+/
K+ pumping capacity, and also high levels of the antioxidant reduced
glutathione, throughout their lifespan. ere are also other dierences
in physiology of dog and cat red cells pertinent to the subject of this
review, and which are considered later.
Dog and Cat Red Cells
In the absence of shear stress, human red cells have the classic
biconcave shape with a diameter of about 8 µm. Dog and cat red
cells have a similar appearance but are somewhat smaller, at 7 µm
and 5.5–6.3 µm, respectively [7]. Cat red cells, in particular, show a
degree of anisocytosis and also tend to lack the central pallor which
is easily recognizable in the more obviously biconcave shape of dog
and human red cells. e oxygen-carrying pigment Hb is found in all
vertebrates with the exception of a few species of Antarctic sh [8].
e latter live at subzero temperatures and thereby survive and carry

John S Gibson (2019) Oxidative stress and haemolytic anaemia in dogs and cats: a comparative approach
Integr J Vet Biosci, Volume 3(3): 2–5, 2019
out aerobic metabolism using only the additional oxygen dissolved
in plasma at these low temperatures. ere are species variations in
Hb, however. In this context, cat Hb is noticeable in having 8–10
readily oxidizable sulphydryl groups [9, 10] whilst most other species
including humans and dogs have only two main ones, represented
by the highly conserved β93 cysteines [11, 12]. Cat Hb also readily
dissociates from the usual tetrameric form to dimers [13] which have
a greater tendency for autoxidation [14]. Heinz bodies, denatured,
precipitated sulphHb, are a special feature of oxidative stress [14].
ey are also found in the circulation of healthy cats, however, at
up to 5–10 % red cells, presumably because of their greater number
of oxidative sites in Hb and impaired red cell antioxidant defence,
together with the poor ability of the non-sinusoidal feline spleen to
remove Heinz body-containing red cells [15]. Cats also have two main
Hbs A and B [9, 16]. HbA is most prevalent in domestic short- and
long-haired cats have HbA (98 %) but a few breeds have greater levels
of HbB (eg 10 % Persians and 14 % in Abyssinians, with as much as 50
% in Devon Rexs) and geographically to occur {eg [17]}. e oxygen
anity of many species is reduced by organic phosphates, especially
2,3-diphosphoglycerate (2,3-DPG or 2,3-biphosphoglycerate), but cat
HbA is less responsive to the reduction in P50 whilst HbB does not
respond at all [18, 19]. Cat red cells also have low levels of 2,3-DPG
[20] which is understandable if it has little regulatory eect on oxygen
anity. Dogs have also several Hbs and more than twelve blood
groups [21] but react like human Hb to 2,3-DPG.
Red Cell Metabolism
Mature red cells lack mitochondria and are therefore dependent
on anaerobic glycolysis for ATP synthesis [3]. Compared with
the citric acid (Kreb cycle) of aerobic respiration this is relatively
inecient, producing two molecules of ATP per glucose moiety
(compared with thirty six in mitochondrial aerobic respiration).
Glycolysis comprises ten enzymatic steps [1], although the main rate
limiting enzymes are hexokinase and pyruvate kinase, at the start and
end of the chain, respectively. In addition to ATP, the pathway also
syntheses reducing power in the form of NADH. NADH is necessary
to reduce methaemoglobin (metHb) using methaemoglobin reductase
(or cytochrome b reductase) – one of the main red cell antioxidant
defences. An o-shoot of the glycolytic pathway called the pentose
phosphate shunt (or hexose monophosphate shunt) is used to make the
reducing compound NADPH, a substrate for glutathione reductase – a
second main antioxidant enzyme – which reduces oxidised glutathione
(GSSG) back to reduced glutathione (GSH). Under normal conditions,
glycolysis uses the majority of glucose metabolised by the red cell, with
the pentose phosphate shunt accounting for only about 10 % of the
ux. Inhibition of the rst enzyme of the pentose phosphate shunt,
glucose-6-phosphate dehydrogenase, by high NADPH / NADP ratios
is responsible and this enzyme normally operates at only a low level of
its maximum capacity. Under conditions of oxidative stress, however,
as NADPH / NADP ratios fall, glucose is preferentially channelled
along the pentose phosphate shunt. Interestingly, deoxyHb which
preferentially binds to the cytoplasmic tail of the anion exchanger (or
Band 3) displaces glycolytic and other enzymes so that deoxygenated
red cells carry out more glycolysis, oxygenated ones produce more
NADPH [22, 23] providing a physiological switch to channel glucose
through one or other pathways. In addition, the red cells of some
species are less permeable to glucose, eg some sh and pigs [1, 24,
25]. In these cases, the pentose phosphate shunt pathways can be
used as an alternative to glycolysis for synthesis of ATP, metabolising
nucleosides, such as inosine and metabolites of ribose, which enter
into the distal part of the glycolytic pathway.
e third red cell metabolic pathway of note is the Rapaport-
Luebering shunt (1950s). is uses the enzyme biphosphoglycerate
mutase to produce 2,3-DPG (2,3-BPG) – apparently conned to cells
of the erythroid lineage and placental cells [26] and accounts for about
20 % of the glucose passing through glycolysis. ere is a metabolic cost
to this, as the Rapaport-Luebering shunt bypasses phosphoglycerate
kinase with the loss of one ATP of the two molecules of ATP from
metabolism of glucose. Congenital enzyme deciencies in the red
cell metabolic pathways have been well described in humans [2, 27].
Some genetic deciencies have also been described in dogs and cats
[Table 1]. Whilst oxidative threat is not the root of these conditions,
a defect in antioxidant defences will accompany the inadequacies
in glucose metabolism which underlie the loss of ATP, and which
represents the main cause of red cell instability.
Table 1. Some inherited causes of haemolytic anaemia in dogs and cats.
Catalase American foxhound, beagle [55]
Hereditary elliptocytosis Band 4,1 deciency [56]
Hereditary spherocytosis Autosomal recessive trait in chondrodysplastic
Alaskan malamute dwarf dogs
Hereditary stomatocytosis Schnauzers [57,59]
Methaemoglobin redutase Dogs (toy Alaskan Eskimo, miniature poodle,
cocker/poodle cross) and cats – domestic short
hair [60,61,62]
Osmotic fragility syndrome Abyssinian, Somali, Siamese and domestic short
hair cats [63–64]
Phosphofructokinase (PFK)
deciency
English springer spaniels, American cocker
spaniels, whippets [65–66]
Pyruvate kinase (PK)
deciency
Basenjis, Cairn terrier, West Highland white
terriers, beagles, cairn terriers, miniature
poodles, dachshunds, Chihuahus, American
Eskimo toy dogs, pugs, American Labrador
retrievers; Abyssinian, Somali and domestic
shorthaired cats [67]
Oxidative Challenge
Red cells are also subject to considerable oxidative stress
throughout their lifespan. Oxidative challenges arise from several
underlying conditions and sources [28, 29]. First, oxygen is potentially
toxic and their function as the main oxygen-carrying cell of the body
exposes them continually to the threat of oxygen damage. Whilst
in other tissues, there is always some slippage of oxygen away from
its mitochondrial function in aerobic respiration, which generates
superoxide anion and other free radicals, in red cells, the iron-
containing Hb is the major source of reactive oxygen species [29,

John S Gibson (2019) Oxidative stress and haemolytic anaemia in dogs and cats: a comparative approach
Integr J Vet Biosci, Volume 3(3): 3–5, 2019
30]. e ferrous Fe2+ in heme groups is potentially unstable and liable
to autoxidation to ferric Fe3+, generating superoxide and, through
dismutation, hydrogen peroxide [31] which may be removed by one
of the important red cell antioxidant enzymes, catalase. Heme iron
is also able to take part in the Fenton and Haber-Weiss reactions to
generate hydroxyl and other free radicals [2, 32]. Red cell NADPH
oxygenase is a further source of endogenous oxidants [28, 33]. Around
0.5–3% red cell haemoglobin is oxidized daily [34], producing a
constant source of methaemoglobin, although levels are usually kept
below 1% through the reducing action of methaemoglobin reductase
[35]. In addition, there is the threat from exogenous oxidants which
may enter the circulation from other tissues, for example following
ischaemia / reperfusion [36], or the action of xanthine oxidase on
hypoxanthine [37] or also via ingested or iatrogenic oxidants [7]. Cat
Hb more susceptible to oxidants (Harvey & Kaneko 1976), especially
feline HbB cf feline HbA. Counterintuitively, dogs with red cells
containing high levels of K+, and also high levels of the antioxidant
reduced glutathione notably Japanese breeds [38] appear more
susceptible to oxidative damage than the more common low K+ ones.
A number of systemic diseases are associated. Some of these include
diabetes mellitus, hepatic problems, hyperthyroidism (especially in
cats), neoplasia, severe hypophosphataemia (eg refeeding syndrome
in cats) and uraemic syndrome.
Oxidative red cell damage from ingestion of products from
Allium species (onions, garlic and related plants – see [39] for a list
of plants) are particularly heavily implicated in the case of dogs and
cats. Onion poisoning in dogs has been recognised since the 1930s
[40] and is due mainly to sulphur-containing organic compounds,
which give the characteristic odour of these foods [39]. ese
compounds are not destroyed by cooking or spoilage. Metabolites
particularly propylsulphides are implicated in onion-induced
oxidant damage of red cells in dogs and cats [41]. Animals probably
need to consume about 0.5 % of their body weight in onions to be
aected [42], though of course the wet weight and the concentration
of the active ingredient will be very variable between feedstus.
Cats are less frequently aected by Allium spp. toxicity because of
their dietary preferences though cases do occur, for example in ill
animals fed on human baby food [43]. Ironically, the same sulphur-
containing organic compounds which cause harm to dogs and cats
are associated with the therapeutic benets of Allium spp. in humans
[44]. Cats also have low hepatic glucuronidation capacity. ey lack
many uridine diphosphate glucuronyltransferases (UGTs) which
makes them particularly susceptible to a number of iatrogenic drugs.
ey thus have a very poor ability to metabolise compounds such
as acetaminophen and salicylic acid [45], for which there is no safe
dose. In both dog and cat, overdoses with acetaminophen leads to the
accumulation of metabolites such as p-aminophenol (PAP) in their red
cells, which lack N-acetyltransferase 2 (NAT2) to remove it. e result
is methaemoglobinaemia [46]. Overdose in other species including
humans, by comparison, is associated with hepatic toxicity induced
by the metabolite N-acetyl-p-benzoquinoneimine (NADPQI) rather
than oxidative damage to red cells. Heavy metals are also implicated
in oxidative damage to red cells, particularly in dogs. Commoner
causes include zinc toxicity (through ingestion of toys, bolts or coins
containing high levels of zinc) [47] or iron overload. e latter is
usually iatrogenic through iron injections or repeat transfusions. Some
other common iatrogenic oxidants and toxins are listed in [Table 2],
with a more complete list is provided in Haematology texts eg [7].
Table 2. Some toxins and iatrogenic oxidants causing haemolytic anaemia in dogs and
cats.
Acetaminophen (paracetamol)
Acetylsalicylic acid (aspirin)
Allium spp.
Benzocaine
Carprofen and other non-steroidal anti-inammatories
Copper
Iron overload
DL-methionine
Methylene blue
Phenylhydrazine
Propylene glycol
Vitamin K and vitamin K antagonists
Zinc
Red Cell Antioxidant Defence
Notwithstanding the potential oxidative peril and their limited
capacity for repair by protein synthesis, red cells must survive for
some one hundred and twenty days in the case of humans and dogs,
and about seventy days in the case of cats. Although the red cell is
well equipped with antioxidant defences, problems arise when
oxidative challenge exceeds the red cell antioxidant capacity. e
result is oxidative damage to membrane lipids and proteins, and to
haemoglobin itself. Oxidised haemoglobin, methaemoglobin (heme
Fe3+ instead of the normal Fe2+), is unable to carry oxygen and is
also liable to denaturation and precipitation as insoluble sulphHb
containing Heinz bodies, or to form eccentrocytes in which the
Hb is restricted to one side of the cell [13]. Other changes include
crosslinking of the cytoskeleton, thiol oxidation, depletion of reduced
glutathione and cation imbalance. e result is a fragile red cells with
impaired rheology liable to intravascular haemolysis with anaemia,
haemoglobinuria and poor oxygen-carrying capacity [48].
Antioxidant provision of red cells is provided by both enzymatic and
non-enzymatic pathways. Five enzymes are heavily involved: catalase
which reduces hydrogen peroxide to oxygen and water, glutathione
reductase uses NADH to reduce oxidised methaemoglobin, superoxide
dismutase scavenges superoxide anions generating hydrogen peroxide
and oxygen in the process, and glutathione peroxidase uses NADPH
to remove both red cell hydrogen peroxide and organic peroxides [49],
as does membrane-associated perioxiredoxin-2 which can be reduced
via reduced glutathione, vitamin C or thioredoxin. Activities of these
enzymes do vary between species [50–52]. Catalase activity in the
red cells of dierence species is very variable [50, 53, 54]. Expression

John S Gibson (2019) Oxidative stress and haemolytic anaemia in dogs and cats: a comparative approach
Integr J Vet Biosci, Volume 3(3): 4–5, 2019
in dog red cells occurs at about a tenth of the amount in humans
whilst its specic activity is around a third that of human catalase
[55]. As a result, overall catalase activity in dog red cells is a thirtieth
that in humans [53, 55]. Non-enzymatic defence includes reduced
glutathione, vitamin C and vitamin E. erapeutic antioxidants
include dosing with N-acetyl cysteine, vitamin C and E. None are
particularly eective for rapid protection [39]. ere is a need for
more ecacious compounds. ese must be eective in the short term
and protect red cells from further oxidative damage and haemolysis
without the requirement for prolonged metabolism. Some human
compounds are listed in [Table 3].
Table 3. Antioxidants used in chemoprophylaxis of sickle cell disease in humans.
Therapy Effect References
Acetyl-L-carnitine Protects red cells from peroxidative
damage and maintains normal shape at
lower oxygen tensions
[68]
N-Acetylcysteine Increases levels of reduced glutathione
and decreases haemolysis
[69,70]
Flavonoids
(quercetin, rutin &
morin
Show inhibitory effect on haemolysis
due to thiol group oxidation
[71]
Glutamine Increases NAD redox potential and
NADH levels
[72,73]
Hydroxyurea Reduces markers of oxidative stress,
decreases lipid peroxidation and
increases level of antioxidant enzymes
[74,75]
Iron chelators:
deferiprone &
deferasirox
Remove iron from the membrane of red
cells, decrease lipid peroxidation and
increase antioxidant capacity
[76,77]
α-lipoic acid Protects red cells from peroxyl radical
induced haemolysis, increases levels
of reduced glutathione and increased
antioxidant gene expression
[78,79]
Melatonin Increases levels of antioxidants and
reduces rate of haemolysis
[80]
Statins Protects against oxidative damage by
increasing nitric oxide metabolites and
C-reactive protein
[81,82]
Vitamin C and E Decreases production of reactive oxygen
species, increases levels of reduced
glutathione and reduces haemolysis
[83]
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Citation:
Gibson JS, Wadud R, Lu D C-Y, Brewin JN, Rees DC (2019) Oxidative stress and haemolytic anaemia in dogs and cats: a comparative approach. Integr J Vet Biosci
Volume 3(3): 1–5.