Clinica Chimica Acta 296 (2000) 1–15

www.elsevier.com / locate / clinchim

Review
Chemical and biological activity of free radical
‘scavengers’ in allergic diseases

´ a , *, Cristina Perez-Gomez
Jose´ M. Mates ´ ´ a
, Miguel Blanca b
a
´
Department of Molecular Biology and Biochemistry, Faculty of Sciences, University of Malaga ,
´
Campus de Teatinos, s /n, 29071 Malaga , Spain
b
´ C, Plaza del Hospital Civil, 29009 Malaga
Allergy Unit of Hospital Carlos Haya, Pabellon ´ , Spain

Received 30 November 1999; received in revised form 3 February 2000; accepted 10 February 2000

Abstract

Reactive oxygen species (ROS) are generated constantly in vivo. They can lead to lipid
peroxidation and oxidation of some enzymes, as well as protein oxidation and degradation. Cells
possess several biological systems, defined as ‘scavengers’, to protect themselves from the
radical-mediated damage. Immune cells may discharge their arsenal of toxic agents against host
tissues, resulting in oxidative damage and inflammation. Therefore, free radical production and
disturbance in redox status can modulate the expression of a variety of immune and inflammatory
molecules, leading to inflammatory processes, both exacerbating inflammation and effecting tissue
damage. Recently, abnormal immunity has been related to oxidative imbalance, and antioxidant
functions are linked to anti-inflammatory and / or immunosuppressive properties. Currently, allergy
is one of the most important human diseases. We studied the role of the primary antioxidant
defence system, constituted by the antioxidant enzymes superoxide dismutase, catalase and
glutathione peroxidase, protecting cells from toxic oxygen. We analyzed how they are involved in
blood cells detoxification, and how the imbalance of reactive oxygen species is related to
inflammation in allergic diseases by affecting immune cells. Finally, we discuss the published data
that relates anti-free radical therapy to the management of human allergic diseases.  2000
Elsevier Science B.V. All rights reserved.

Abbreviations: CAT, catalase (EC 1.11.1.6); EPO, eosinophil peroxidase (EC 1.11.1.2); FMLP, N-formyl-
methionyl-leucyl-phenylalanine; GM-CSF, granulocyte-macrophage colony-stimulating factor; GPX, gluta-
thione peroxidase (EC 1.11.1.19); GSH, glutathione; IFN-a, interferon alpha; IFN-g, interferon gamma; IgE,
immunoglobulin E; NADPH, reduced nicotinamide-adenine dinucleotide phosphate; NFkB, nuclear factor
kappa-B; NOS: nitric oxide synthase (1.14.13.39); PAF, platelet-activating factor; PMA, phorbol 12-myristate
13-acetate; PMNs, polymophonuclear leukocytes; ROS, reactive oxygen species; SOD, superoxide dismutase
(EC 1.15.1.1); TNF-a, tumor necrosis factor alpha
*Corresponding author. Tel.: 134-95-213-1930; fax: 134-95-213-2000.
E-mail address: jmates@uma.es (J.M. Mates)´

0009-8981 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved.
PII: S0009-8981( 00 )00215-1
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J.M. Mates

Keywords: Allergy; Catalase; Glutathione peroxidase; Oxidative stress; Reactive oxygen species;
Superoxide dismutase

1. Introduction

The principal physiological function of the immune system is the removal of

infectious agents. In this process, cells can be damaged. Therefore, the organism
has evolved different ways of eliminating actively its own potentially harmful
products, so that the host may survive. Potentially injurious immune reactions
may be prevented either by inactivating functionally the responding lympho-
cytes, or by eliminating the harmful radical reaction products [1]. Cells possess
different systems, defined as ‘scavengers’, to protect themselves from the
radical-mediated damage and, among these, some enzymes or chemical struc-
tures have the role of antioxidants.
Reactive oxygen species (ROS), including hydroxyl radicals ( ? OH), superox-
ide anion (O ?2
2 ), hydrogen peroxide (H 2 O 2 ) and nitric oxide (NO) are very
transient species due to their high chemical reactivity that leads to lipid
peroxidation and oxidation of some enzymes, and a massive protein oxidation
and degradation [2,3].
Neutrophils constitute 60 per cent of the circulating leukocytes and they are
the most abundant cellular components of the immune system. They may
discharge their arsenal of toxic agents against host tissues, resulting in oxidative
damage and inflammation. The combination of phagocytosis of bacteria and
secretion of proteolytic enzymes and immuno-modulatory compounds that assist
in the killing and digestion of bacteria, are accompanied by respiratory burst,
involving a sudden stimulus-induced increase in non-mitochondrial oxidative
metabolism which results in the production of ROS [4].
ROS generation through normal cellular metabolism and by exogenous insults
is a constant problem for which cells have developed multiple protective
mechanisms to survive [5,6]. ROS are mainly generated by mitochondria at the
portion of the electron transport chain as the toxic by-products of oxidative
phosphorylation, their energy generating pathway [7]. The interruption of
oxidative phosphorylation results in decreased levels of ATP. Whatever the
cause(s) and sequence of events are, respiratory chain deficiencies appear to play
an important role in the pathogenesis of many diseases [8]. In addition, free
radical production and disturbance in redox status can modulate the expression
of a variety of immune and inflammatory molecules [9–11], leading to
inflammatory processes, both exacerbating inflammation and effecting tissue
damage [12]. Recently, it has been suggested that abnormal immunity has been
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J.M. Mates 3

related to oxidative imbalance [13–17], and antioxidant functions are linked to

anti-inflammatory and / or immunosuppressive properties [18–20].
Since tissue injury and inflammation lead to increased oxidative stress, it
seems logical that good antioxidant status might diminish tissue damage in
allergic diseases [6–8]. Herein, we will see the role of the primary antioxidant
defence system which is constituted by the antioxidant enzymes, protecting cells
from toxic oxygen. We will analyze how they are involved in blood cells
detoxification, and how the imbalance of reactive oxygen species is related to
inflammation in allergic diseases by affecting immune cells. A further in-
vestigation in antioxidant enzymes may open a new pathway in the knowledge
of allergic diseases.

2. Reactive oxygen species and free radical scavengers

Generation of hydrogen peroxide takes place through the dismutation of

superoxide. Therefore any biological system generating O ?2 2 will produce H 2 O 2 .
However, there are enzymes localized in peroxisomes that produce H 2 O 2
without intermediation of O 2?2 . Contrary to O 2?2 , H 2 O 2 is able to cross cell
membranes and inside the cells it can react with Fe 21 or Cu 1 to form hydroxyl
radicals via the Fenton reaction [21]:

Fe 21 1 H 2 O → Fe 31 1 ? OH 1 2 OH (Fenton reaction)

O 2?2 1 H 2 O 2 → O 2 1 ? OH 1 2 OH (Haber–Weiss reaction)

The metal-catalysed Haber–Weiss reaction may involve the participation of

either free iron (or copper) or iron sequestered in the form of nucleotide iron
complexes, ferritin, lactoferrin, hemoglobin, and myoglobin.
The wide array of enzymatic and non-enzymatic antioxidant defences includes
superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT),
ascorbic acid (vitamin C), a-tocopherol (vitamin E), glutathione (GSH), beta-
carotene, vitamin A, NADPH, adenosine, coenzyme Q, urate, methionine,
cysteine, phenols and flavonoids [22,23].
Superoxide dismutase (EC 1.15.1.1) destroys the highly reactive radical
superoxide by conversion into the less reactive peroxide (H 2 O 2 ), that can be
destroyed by catalase or glutathione peroxidase reactions [21]:
SOD
O2 1 O2 1 2 H → H2O2 1 O2

Catalase (EC 1.11.1.6) is a highly reactive enzyme, reacting with H 2 O 2 to

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J.M. Mates

form water and molecular oxygen; and with hydrogen donors such as methanol,
ethanol, formic acid or phenols [22]:
CAT
2H 2 O 2 → H 2 O 1 O 2

CAT
ROOH 1 AH 2 → H 2 O 1 ROH 1 A

Glutathione peroxidase (EC 1.11.1.19) catalyses the reduction of a variety of

hydroperoxides (ROOH and H 2 O 2 ) using GSH, thereby protecting mammalian
cells against oxidative damage and, reducing, among others, cellular lipid
hydroperoxides [22]:
GPX
ROOH 1 2 GSH → ROH 1 GSSG 1 H 2 O

The flavin containing thioredoxin reductase (EC 1.6.4.5) is an ubiquitous

enzyme able to reduce O ?2 2 and NO by using thioredoxin as a substrate [23].
Transferrin and ferritin sequester iron ions, while ceruloplasmin sequesters
copper ions so that these ions are not available to catalyse the Haber–Weiss
reaction generating ? OH or to perform the decomposition of hydroperoxides.
Ceruloplasmin has also ferroxidase activity: it oxidizes Fe 21 to Fe 31 and so
inhibits ? OH formation from H 2 O 2 and iron dependent lipoperoxidation [24].
a-Tocopherol is concentrated inside the membranes, in blood lipoproteins and
adrenal glands. It quenches and reacts with 1 O 2 and is a scavenger of ? OH, able
to protect membranes from these extremely reactive species. However, its major
antioxidant action in biological membranes is to act as a chain breaking
antioxidant, donating labile hydrogen to peroxy and alkoxy radicals, thereby
breaking the radical chain. It has been proposed that a-TO ? may be reduced by
ascorbic acid or reduced glutathione. These are scavengers of ROS and other
reactive free radicals. b-Carotene is a powerful scavenger of 1 O 2 . Urate binds
iron and copper and scavenges ? OH, 1 O 2 and peroxy radicals [25].
Animal studies have shown that circulating cells exhibit membrane alterations
when there is a lack of vitamin E in the diet. In fact, the mitochondrial
membranes of the reticulocytes and lymphocytes appear bloated and dis-
integrated; the number of platelets, which are more adhesive, increases and
produces more thromboxanes in comparison with those of normal rats. The
response of T and B lymphocytes to mitogenic stimuli, the mixed lymphocyte
reaction and the number of cells forming plates are considerably depressed. On
the contrary, carotenoids and in particular beta-carotene either directly or
indirectly as precursor of vitamin A, enhance T cell proliferation and cytotoxici-
ty, macrophage killing, and TNF-a secretion [24].
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3. ROS generation and histamine and nitric oxide release

All allergic diseases are characterized by a specific pattern of inflammation

that is largely driven by IgE-dependent mechanisms. Although allergen avoid-
ance is, by far, the most effective way against allergic reaction, immunotherapy
is the only clinical treatment to effectively prevent the allergic manifestations.
Not only does Immunotherapy act only producing blocking antibodies, but it
also favors the transformation of lymphocytes T helper 2 (Th 2 ) into Th 1 . At the
same time, the production of interferon gamma (IFN-g) increases and there is an
inhibition in the production of IgE by B lymphocytes (Fig. 1). In these processes
stimulated cells generate a considerable amount of reactive oxygen species.
Thus, we will describe antioxidant enzymes appearing to work with immuno-
therapy in order to effect some immune-like functions against inflammation and
allergic manifestations (Fig. 2).
Mast cells exist throughout most tissues although they are more prevalent in
areas that come into contact with the external environment, such as the skin,

Fig. 1. Immunotherapy favors the transformation of lymphocytes Th 2 into lymphocytes Th 1 . By

avoiding this transformation, IFN-a production and inhibition of B lymphocytes that secrete IgE,
is achieved. The alternative pathways for lymphocytes Th 2 transformation and some of the
cytokines involved are shown.
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J.M. Mates

Fig. 2. Antioxidant defence might block histamine (and other chemicals and molecules) released
from mast cell. The production of ROS has been detected in cells stimulated with cytokines such
as transforming growth factor-b (TGF-b), interleukins (IL-x), granulocyte macrophage colony
stimulating factor (GM-CSF) and tumor necrosis factor-a (TNF-a) with peptide growth factors
such as platelet derived growth factor (PDGF) and basic fibroblast growth factor (BFGF), with
agonists of receptors such as angiotensin II (AGII) and lysophosphatidic acid (LPA), or with drugs
as phorbol 12-myristate 13-acetate (PMA).

lungs, and gastrointestinal tract. They have a recognized pathophysiologic role

as effector cells in immediate hypersensitivity reactions [24]. Such mast cells,
when activated by either immunologic or non-immunologic stimuli, release and
generate chemical mediators such as histamine, protaglandins, bradykinins,
platelet activating factors (PAF) and leukotrienes which then act on surrounding
tissues (Fig. 2).
Hydrogen peroxide stimulates histamine release. It is reduced by addition of
catalase and flavonoids, showing a relationship and a balance among antioxidant
enzymes, active oxygen and histamine release [26].
IFN-a and IFN-g enhance monocyte-mediated activity. IFN-a directly
activates blood monocyte superoxide anion release in allergic patients [27].
Superoxide generation correlates with the degree of bronchial hyperresponsive-
ness to inhaled histamine. These results hint that polymorphonuclear leukocytes
(PMNs) in asthmatic children, especially in those with attacks, generate more
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J.M. Mates 7

active oxygen species than those in control subjects, and that airway inflamma-
tion caused by O ?2 2 may be closely related to bronchial hyperresponsiveness
[28].
During phagocytosis, neutrophilic and eosinophilic granulocytes undergo
metabolic activation and degranulation. This leads to accumulation of H 2 O 2 in
the extracellular environment in inflammation with release of histamine. This
release reaction was inhibited by azide and catalase [29].
On the other hand, asthmatic patients show an increased expression of
inducible nitric oxide synthase (iNOS) in airway epithelial cells and an
increased level of NO in exhaled air. The NO derived from airway epithelial
cells may be a mechanism for amplifying and perpetuating asthmatic inflamma-
tion, through inhibition of Th 1 cells and their production of IFN-g. This would
result in an increase in the number of Th 2 cells and the cytokines IL-4 and IL-5.
Thus, it has been argued that the development of specific iNOS inhibitors may
also represent a novel therapeutic approach for asthma and other allergic
diseases [30].
Another issue is the molecular mechanisms of ROS in affecting these immuno
and inflammatory cell activities. It can be driven by secondary cytokine release.
More information on the effects at the transcriptional level, such as on NF-kB
are needed [31]. This might be a clinically relevant pathway in the case of
exposure to pollutants or respiratory tract infections, and it seems to occur in
bronchial epithelial cells [32].

4. Free radical scavengers and blood detoxification

Neutrophil populations have the innate property of generating highly reactive

oxygen free radical species such as O ?2 ?
2 , HO and H 2 O 2 , and they produce them
during inflammatory reactions [33]. CAT, but not SOD, prevented neutrophil
activation measured as (1) induction of cytotoxic responses, (2) increase of
neutrophil adhesiveness to cell-free surfaces, and (3) inhibition of chemotactic
responses to N-formyl-methionyl-leucyl-phenylalanine (FMLP). These findings
suggest that H 2 O 2 may play a major role in neutrophil activation [34].
When neutrophils were blocked from direct attachment to endothelial cells,
endothelial cell damage was ameliorated. SOD partially inhibited this endotheli-
al cell injury [35]. The respiratory burst is a coordinated series of metabolic
events that take place when PMNs and other phagocytes are exposed to an
appropiate stimulus such as phorbol 12-myristate 13-acetate (PMA), the
chemotactic peptide FMLP or leukotriene B4. In fact, stimulation of PMNs
rapidly increases oxygen consumption and produces superoxide [36].
Ig-stimulated eosinophils can also induce damage to respiratory epithelium.
Granule basic proteins and reactive oxygen radicals may be responsible for the
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J.M. Mates

injury. Peripheral blood eosinophils and neutrophils obtained from allergic

patients, and stimulated with PMA, displayed a significantly higher superoxide
generation [37]. CAT inhibited partially the appearance of irregularities on the
surface of the epithelium. Moreover, the addition of either CAT or SOD reduced
partly the activity of PMA-stimulated eosinophils [38].
GPX also has a protective role against oxidative stress and a regulatory
function in arachidonic acid metabolism, which leads to prostaglandins synthesis
[39]. Reduced GPX activity contributes to an allergic inflammation and it is
associated with intolerance to some food and aspirin (Table 1). It seems likely
that the reduced activity of this enzyme in platelets and blood may reflect
mechanisms associated with the pathogenesis and severity of allergic disorders
[47]. The lowest levels of GPX activity were found during the acute attack,
which were significantly less than the control, even in patients with mild
symptomatology [48].
SOD is constitutively expressed in leukocytes and other tissues. Significant
induction of SOD activity occurs in peripheral blood leukocytes incubated in
vitro with paraquat, an agent known to cause intracellular superoxide production
[49]. Cu,Zn-SOD activities in platelets from the asthmatics were significantly
higher than those from normal healthy subjects. Platelet Cu,Zn-SOD activities
were significantly higher in atopic than in non-atopic asthmatics [50]. These
results suggest that an oxidant-to-antioxidant imbalance may play a role in the
inflammatory situations in the airways of asthmatics.
In fact, an imbalance between oxidants and antioxidants is proposed in

Table 1
Allergy and main antioxidant enzymes involved in the allergic response
Clinical manifestations Allergens Enzymes Reference
Hypersensitivity reactions Skin tests SOD [21]
Hypersensitivity reactions Olive pollen SOD [40]
Hypersensitivity reactions Pollens and house dust mite SOD/ CAT [41]
Hypersensitivity reactions Latex SOD [42]
Acute respiratory syndrome Ragweed SOD [43]
Asthma Anti-asthma drugs SOD/ GPX [28]
Asthma Anti-asthma drugs EPO a [22]
Asthma Oxidants SOD [44]
Asthma Mercury GPX [45]
Asthma Foods GPX [21]
Asthma Aspirin GPX [22]
Asthma Non aspirin, non NSAID b GPX [46]
Allergic encephalomyelitis Myelin protein NOS c [21]
a
EPO: Eosinophil peroxidase.
b
NSAID: non-steroidal anti-inflammatory drugs.
c
NOS: nitric oxide synthase.
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J.M. Mates 9

smokers and in patients with airways diseases. This hypothesis has been tested
by measuring the Trolox equivalent antioxidant capacity (TEAC) of plasma and
the concentrations of lipid peroxidation products as indices of overall oxidative
stress. The plasma TEAC was markedly reduced, with increased levels of lipid
peroxidation products in healthy chronic smokers as compared with healthy
nonsmokers. Plasma TEAC was also low in patients presenting with acute
exacerbations of chronic obstructive pulmonary disease (COPD) or asthma [51].
IgE-mediated systemic anaphylaxis can also follow parenteral SOD adminis-
tration [52]. In addition, olive pollen extracts have a heterogeneous composition,
with several important allergens, one of which showed a high degree of
homology with a SOD. Otherwise, characterization and identification of latex
allergens show that some of the IgE-reactive protein spots have high homology
with SOD (Table 1).
Cytokines are implicated in allergic diseases and can modulate effector
functions of eosinophils stimulated by another agonist, inducing eosinophil
degranulation and superoxide production in vitro. Therefore these cytokines may
be important in the release of toxic granule proteins from eosinophils in allergic
diseases [53].
Indeed, asthma is characterized by an accumulation of activated eosinophils in
the airway. Bronchial epithelial cells have been shown to release cytokines
including granulocyte-macrophage colony-stimulating factor (GM-CSF). Eosin-
ophil peroxidase (EPO) stimulates epithelial cells to release GM-CSF and forms
a self-stimulatory cycle [54]. On the other hand, in bronchial asthma, in-
flammatory cells, infiltrating the airway mucosa, release oxygen radicals that
cause tissue damage and amplify the airway inflammation. Therefore, anti-
oxidant enzymes may have a protective effect on the airways, and a beneficial
effect on bronchial asthma [43]. In addition, oxidative stress can be measured
rather easily and non invasively by hydrogen peroxide, pentane, CO and NO in
exhaled air [55–59].

5. Antioxidant enzymes also against cytotoxicity?

Cell-mediated cytotoxicity is a kind of immune response mainly due to T and

NK cells. Examples of such reactions are the T cell infiltration of tumor beds
and the T cell infiltration of blood vessels and alveoli [60]. Oxygen radicals have
been detected in a variety of stimulated blood cells (Fig. 2) [61]. For instance,
during various biological processes as inflammation or septic shock, free radical
damages are not only caused by a direct generation of oxygen radicals by
phagocytes, but also by a TNF-a-mediated generation in target cells. The
oxidative effect of TNF-a is beneficial in physiological conditions as it can
destroy cancerous or virus infested cells. But this effect can be deleterious in a
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J.M. Mates

situation of deficiency in some antioxidants. TNF-a-induced free radicals can

increase the replication of virus as HIV-1 and destroy immunocompetent cells
such as T cells. This last action explains the defect in cellular immunity
observed in oxidative stress and the immunostimulatory effect of many
antioxidants. In this event, catalase has the better protecting effect whereas
Cu,Zn-SOD has little effect [62]. So, antioxidants have been demonstrated as
protective against TNF-a cytotoxicity.
On the other hand, addition of IFN-a or IFN-g enhanced the monocyte-
mediated cytotoxicity of a colon carcinoma cell line. However, inhibitors of
H 2 O 2 -myeloperoxidase system suppressed both IFN-a- and IFN-g-induced
cytotoxicity of these cells [63].

6. Summary and prospects

There is considerable interest in the therapeutic use of antioxidants. This may

involve the use of naturally occurring antioxidants or completely synthetic
molecules. In addition, there is evidence that some drugs already used clinically
may exert part or all their effect by antioxidant mechanisms. Infusions of SOD
in liposomes (usually with catalase) have been reported to protect animals
against O 2 toxicity. Cu,Zn-SOD has an anti-inflammatory effect in animal
models of acute inflammation, in part because it can decrease the number of
neutrophils entering sites of inflammation. A wide variety of Cu,Zn-SOD
conjugates are available, including polyethylene glycol (PEG)-SOD, Ficoll-
SOD, lecithinized SOD, polyamine conjugated SOD, cationized SOD, ge-
netically engineered SOD polymers, pyran-SOD and albumin-SOD complexes.
All have longer circulating half-lives than the unconjugated SOD molecules
[25]. In addition, several low-molecular-mass compounds that react with
superoxide anion have been described. Most contain transition-metal ions.
Examples include iron porphyrins, a complex of manganese ions with the
chelatins agents desferrioxamine and copper ions chelated to amino acids or to
anti-inflammatory drugs [25,64].
It is well established that immunotherapy can prevent or reduce allergy both
increasing IFN-g production and decreasing IgE production. Nevertheless,
sometimes immunotherapy is not the best indicated treatment. Drugs, traditional-
ly used against clinical manifestations of allergy, include coumarins, glucocor-
ticoids, anti-leukotrienes and anti-histaminics (Fig. 3).
We know today that immune cell response to allergens induces an accumula-
tion of ROS that brings about mast cell in order to release histamine, the main
chemical mediator of inflammation in allergy. Additionally, active oxygen
becomes toxic when an imbalance arises between antioxidant enzymes and free
radical production during an allergic reaction. Consequently, could the in-
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J.M. Mates 11

Fig. 3. ROS involvement in allergic response and alternative treatments. Alternative treatment to
patients suffering from an allergic process, includes allergen avoidance, immunotherapy, glucocor-
ticoids, coumarins, anti-histaminics and anti-leukotriens. Antioxidant defence system (antioxidant
enzymes and non enzymatic antioxidant substrates) may provide a valuable help for immuno-
therapy and traditional drug. Mast cell (chemical mediators) and eosinophil (granule proteins)
degranulation are also shown. Transformations with dotted arrows represent ROS generation.

vestigation in antioxidant enzymes open a new and complementary pathway in

the treatment of allergic diseases?
We believe that the established connections between antioxidant enzymes and
several allergic diseases (Table 1) may be a sign of their functional importance
in the mechanism of the immunopathologic reaction named allergy. In fact,
oxidative stress and antioxidant enzymes have been widely studied in many
other pathologies and they have also been determined separately as an indicator
of oxidative damage in many patients suffering from an allergic reaction. For
instance, unsaturated lipid components of membrane are essential for a correct
immune function, and represent one of the main targets of free radical damage.
However, a complete study in this field has not been described to date for
allergic patients.
This work provides further clues to the interaction between oxidative stress
and allergic diseases. Therefore, in spite of considering potential negative
consequences of antioxidant therapy, efforts are necessary to fully understand
the relevance of ROS and free radical scavengers in the immune system and
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J.M. Mates

allergy: without any doubt one of the most important human diseases at the
beginning of this new century.

Acknowledgements

We wish to thank Prof. I. Nunez´ ˜ de Castro, Dr. J.A. Segura and Dr. J.C.
Aledo for critically reading the manuscript, to Ms. M. Asenjo, M.D. for her
helpful suggestions, and to Miss C. Segura and Mr. N. McVeigh for the revision
of the spelling and the grammar. This work was supported by projects SAF98-
0150 and SAF98-0153.

References

[1] Dimmeler S, Haendeler J, Sause A, Zeiher AM. Nitric oxide inhibits APO-1 / Fas-mediated
cell death. Cell Growth Differ 1998;9:415–22.
[2] Stamler JS, Hausladen A. Oxidative modifications in nitrosative stress. Nat Struct Biol
1998;5:247–9.
[3] Jourd’heuil D, Mills L, Miles AM, Grisham MB. Effect of nitric oxide on hemoprotein-
catalyzed oxidative reactions. Nitric Oxide 1998;2:37–44.
[4] Pithon Curi TC, De Melo MP, Palanch AC, Miyasaka CK, Curi R. Percentage of
phagocytosis, production of O ?22 , H 2 O 2 and NO, and antioxidant enzyme activities of rat
neutrophils in culture. Cell Biochem Funct 1998;16:43–9.
[5] Ha HC, Sirisoma NS, Kuppusamy P, Zweier JL, Woster PM, Casero Jr RA. The natural
polyamine spermine functions directly as a free radical scavenger. Proc Natl Acad Sci
1998;95:11140–5.
[6] Halliwell B. Vitamin C: poison, prophylactic or panacea? Trends Biochem Sci 1999;24:255–
9.
[7] Melov S, Schneider JA, Day BJ et al. A novel neurological phenotype in mice lacking
mitochondrial manganese superoxide dismutase. Nat Genet 1998;18:159–63.
[8] Schapira AH. Mitochondrial involvement in Parkinson’s disease, Huntington’s disease,
hereditary spastic paraplegia and Friedreich’s ataxia. Biochim Biophys Acta 1999;1410:159–
70.
[9] Sundaresan M, Yu ZX, Ferrans VJ, Irani K, Finkel T. Requeriment for generation of H 2 O 2
for Platelet-Derived-Growth Factor signal transduction. Science 1995;270:296–9.
[10] Kaouass M, Deloyer P, Gouders I, Peulen O, Dandrifosse G. Role of interleukin-1 beta,
interleukin-6, and TNF-alpha in intestinal maturation induced by dietary spermine in rats.
Endocrine 1997;6:187–94.
[11] Kagaya K, Miyakawa Y, Watanabe K, Fukazawa Y. Antigenic role of stress-induced catalase
of Salmonella typhimurium in cell-mediated immunity. Infect Immun 1992;60:1820–5.
[12] Tsai KJ, Hung IJ, Chow CK, Stern A, Chao SS, Chiu DTY. Impaired production of nitric
oxide, superoxide, and hydrogen peroxide in glucose 6-phosphate-dehydrogenase-deficient
granulocytes. FEBS Lett 1998;436:411–4.
[13] Chen C, Zhou J, Xu H, Jiang Y, Zhu G. Effect of selenium suplementation on mice infected
with LP-BM5 MuLV, a murine AIDS model. Biol Trace Elem Res 1997;59:187–93.
 ´ et al. / Clinica Chimica Acta 296 (2000) 1 – 15
J.M. Mates 13

[14] Galan P, Preziosi P, Monget AL et al. Effects of trace element and / or vitamin supple-
mentation on vitamin and mineral status, free radical metabolism and immunological markers
in elderly long-hospitalized subjects. Int J Vitamin Nutr Res 1997;67:450–60.
[15] Zager RA, Burkhart K. Decreased expression of mitochondrial-derived H 2 O 2 and hydroxyl
radical in cytoresistant proximal tubules. Kidney Int 1997;52:942–52.
[16] Battistoni A, Folcarelli S, Cervoni L et al. Role of the dimeric Structure in Cu Zn superoxide
dismutase. pH-dependent, reversible denaturation of the monomeric enzyme from Es-
cherichia coli. J Biol Chem 1998;273:5655–61.
[17] Kiningham RB. Physical activity and the primary prevention of cancer. Prim Care
1998;25:515–36.
[18] De Waart FG, Portengen L, Doekes G, Verwaal CJ, Kok FJ. Effects of 3 months vitamin E
supplementation on indices of the cellular and humoral immune response in elderly subjects.
Br J Nutr 1997;78:761–74.
[19] Chen F, Lu Y, Demers LM, Rojanasakul Y, Shi X, Vallyathan V, Castranova V. Role of
hydroxyl radical in silica-induced NF-kappa B activation in macrophages. Ann Clin Lab Sci
1998;28:1–13.
[20] Shankar AH, Prasad AS. Zinc and immune function: the biological basis of altered resistance
to infection. Am J Clin Nutr 1998;68:447S–63S.
[21] Mates´ JM, Perez-Gomez
´ ´ ´ ˜ de Castro I. Antioxidant enzymes and human diseases.
C. Nunez
Clin Biochem 1999;32:595–603.
[22] Mates´ JM, Sanchez-Jimenez
´ ´ F. Antioxidant enzymes and their implications in
pathophysiologic processes. Front Biosci 1999;4:339–45.
[23] Watson PS, Scalia GM, Galbraith A, Burstow DJ, Bett N, Aroney CN. Lack of effect of
coenzyme Q on left ventricular function in patients with congestive heart failure. J Am Coll
Cardiol 1999;33:1549–52.
[24] Picardo M, Passi S. Free radicals. In: Bos JD, editor, Skin Immune System (SIS), Boca
Raton, New York: CRC Press, 1997, pp. 207–26.
[25] Halliwell B, Gutteridge JMC. Free radicals in Biology and Medicine, New York: Oxford
University Press, 1999.
[26] Ogasawara H, Fujitani T, Drzewiecki G, Middleton Jr. E. The role of hydrogen peroxide in
basophil histamine release and the effect of selected flavonoids. J Allergy Clin Immunol
1986;78:321–8.
[27] Demoly P, Damon M, Michel FB, Godard P. IFN-gamma activates superoxide anion
production in blood monocytes from allergic asthmatic patients. Ann Allergy Astma
Immunol 1995;75:162–6.
[28] Kato M, Morikawa A, Kimura H, Shimizu T, Nakano M, Kuroume T. Effects of antiasthma
drugs on superoxide anion generation from human polymorphonuclear leukocytes or
hypoxanthine-xanthine oxidase system. Int Arch Allergy Appl Immunol 1991;96:128–33.
[29] Stendahl O, Molin L, Lindroth M. Granulocyte-mediated release of histamine from mast
cells. Effect of myeloperoxidase and its inhibition by antiinflammatory sulfone compounds.
Int Arch Allergy Appl Immunol 1983;70:277–84.
[30] Barnes PJ, Liew FY. Nitric oxide and asthmatic inflammation. Immunol Today 1995;16:128–
30.
[31] Barnes PJ, Adcock IM. NF-kappa B: a pivotal role in asthma and a new target for therapy.
Trends Pharmacol Sci 1997;18:46–50.
[32] Barnes PJ, Karin M. Nuclear factor-kappaB: a pivotal transcription factor in chronic
inflammatory diseases. N Eng J Med 1997;66:1066–71.
[33] Rollet-Labelle E, Grange MJ, Elbim C, Marquetty C, Gougerot-Pocidalo MA, Pasquier C.
Hydroxyl radicals as a potential intracellular mediator of polymorphonuclear neutrophil
apoptosis. Free Rad Biol Med 1998;24:563–72.
14 ´ et al. / Clinica Chimica Acta 296 (2000) 1 – 15
J.M. Mates

[34] Geffner JR, Fontan ´ PA, Sordelli DO, Isturiz MA. Neutrophil erythrotoxicity induced by
phorbol myristate acetate: mechanisms involved in neutrophil activation. J Leukoc Biol
1991;49:352–9.
[35] Furuno T, Mitsuyama T, Hidaka K, Tanaka T, Hara N. The role of neutrophil elastase in
human pulmonary artery endothelial cell injury. Int Arch Allergy Immunol 1997;112:262–9.
[36] O’Dowd Y, Newsholme P. Regulation of superoxide production in human polymorphonu-
clear leukocites. Biochem Soc Trans 1997;25:366S.
[37] Schauer U, Leinhaas C, Jager¨ R, Rieger CH. Enhanced superoxide generation by eosinophils
from asthmatic children. Int Arch Allergy Appl Immunol 1991;96:317–21.
[38] Hirata A, Motojima S, Fukuda T, Makino S. Damage to respiratory epithelium by guinea-pig
eosinophils stimulated with IgG-coated Sepharose beads. Clin Exp Allergy 1996;26:848–58.
[39] Hasselmark L, Malmgren R, Zetterstrom ¨ O, Unge G. Selenium supplementation in intrinsic
asthma. Allergy 1993;48:30–6.
´
[40] Boluda L, Alonso C, Fernandez-Caldas E. Purification, characterization, and partial sequenc-
ing of two new allergens of Olea europaea. J Allergy Clin Immunol 1998;101:210–6.
[41] Mates´ JM, Segura JM, Perez-Gomez
´ ´ ´
C, Rosado R, Olalla L, Blanca M, Sanchez-Jimenez ´ F.
Antioxidant enzymatic activities in human blood cells after an allergic reaction to pollen or
house dust mite. Blood Cell Mol Dis 1999;25:103–12.
[42] Posch A, Chen Z, Wheeler C, Dunn MJ, Raulf-Heimsoth M, Baur X. Characterization and
identification of latex allergens by two-dimensional electrophoresis and protein mi-
crosequencing. J Allergy Clin Immunol 1997;99:385–95.
[43] Assa’ad AH, Ballard ET, Sebastian KD, Loven DP, Boivin GP, Lierl MB. Effect of
superoxide dismutase on a rabbit model of chronic allergic asthma. Ann Allergy Astma
Immunol 1998;80:215–24.
[44] Shull S, Heintz NH, Periasamy M et al. Differential regulation of antioxidant enzymes in
response to oxidants. J Biol Chem 1991;266:24398–403.
¨
[45] Bjorkman L, Langworth S, Lind B, Elinder CG, Nordberg M. Activity of antioxidative
enzymes in erythrocytes and concentration of selenium in plasma related to mercury
exposure. J Trace Elem Electrolytes Health Dis 1993;7:157–64.
[46] Misso NL, Powers KA, Gillon RL, Stewart GA, Thompson PJ. Reduced platelet glutathione
peroxidase activity and serum selenium concentration in atopic asthmatic patients. Clin Exp
Allergy 1996;26:838–47.
[47] Bibi H, Schlesinger M, Tabachnik E, Schwartz Y, Iscovitz H, Iaina A. Erythrocyte
glutathione peroxidase activity in asthmatic children. Ann Allergy 1988;61:339–40.
[48] Beasley R, Thomson C, Pearce N. Selenium, glutathione peroxidase and asthma. Clin Exp
Allergy 1991;21:157–9.
[49] Niwa Y, Ishimoto K, Kanoh T. Induction of superoxide dismutase in leukocytes by paraquat:
correlation with age and possible predictor of longevity. Blood 1990;76:835–41.
[50] Malmgrem R, Unge G, Zetterstrom ¨ O, Theorell H, De Wahl K. Lowered glutathione-
peroxidase activity in asthmatic patients with food and aspirin intolerance. Allergy
1986;41:43–5.
[51] Rahman I, Morrison D, Donaldson K, MacNee W. Systemic oxidative stress in asthma,
COPD, and smokers. Am J Respir Crit Care Med 1996;154:1055–60.
[52] Joral A, Boyano T, Mira J, Agud JL, Saiz F. Systemic anaphylaxis following parenteral
orgotein administration. J Investig Allergol Clin Immunol 1993;3:103–4.
[53] Horie S, Gleich GJ, Kita H. Cytokines directly induce degranulation and superoxide
production from human eosinophils. J Allergy Clin Immunol 1996;98:371–81.
[54] Motojima S, Adachi T, Manaka K, Arima M, Fukuda T, Makino S. Eosinophil peroxidase
stimulates the release of granulocyte-macrophage colony-stimulating factor from bronchial
epithelial cells. J Allergy Clin Immunol 1996;98:S216–23.
 ´ et al. / Clinica Chimica Acta 296 (2000) 1 – 15
J.M. Mates 15

[55] Dohlman AW, Black HR, Royall JA. Expired breath hydrogen peroxide is a marker of acute
airway inflammation in pediatric patients with asthma. Am Rev Respir Dis 1993;148:955–
60.
[56] Jobsis Q, Raatgeep HC, Hermans PW, de Jongste JC. Hydrogen peroxide in exhaled air is
increased in stable asthmatic children. Eur Respir J 1997;10:519–21.
[57] Loukides S, Horvath I, Wodehouse T, Cole PJ, Barnes PJ. Elevated levels of expired breath
hydrogen peroxide in bronchiectasis. Am J Respir Crit Care Med 1998;158:991–4.
[58] Horvath I, Donnelly LE, Kiss A, Paredi P, Kharitonov SA, Barnes PJ. Raised levels of
exhaled carbon monoxide are associated with an increased expression of heme oxygenase-1
in airway macrophages in asthma: a new marker of oxidative stress. Thorax 1998;53:668–72.
[59] Paredi P, Kharitonov SA, Loukides S, Pantelidis P, du-Bois RM, Barnes PJ. Exhaled nitric
oxide is increased in active fibrosing alveolitis. Chest 1999;115:1352–6.
[60] Shearer WT, Fleisher TA. The immune system. In: Middleton Jr E, Reed CE, Ellis EF,
Adkinson Jr NF, Yunginger JW, Busse WW, editors, Allergy. Principles and Practice, Vol. I,
St. Louis, Missouri: Mosby, 1998, pp. 1–13.
[61] Barnes PJ. Pathophysiology of allergic inflammation. In: Middleton Jr E, Reed CE, Ellis EF,
Adkinson Jr NF, Yunginger JW, Busse WW, editors, Allergy. Principles and Practice, Vol. I,
St. Louis, Missouri: Mosby, 1998, pp. 356–65.
[62] Ferlat S, Favier A. Tumor necrosis factor (TNF) and oxygen free radicals: potential effects
for immunity. CR Seances Soc Biol Fil 1993;187:296–307.
[63] Webb DS, Gerrard TL. IFN-alpha and IFN-gamma can affect both monocytes and tumor
cells to modulate monocyte-mediated cytotoxicity. J Immunol 1990;144:3643–8.
[64] Salvemini D, Wang Z, Zweier JL et al. A nonpeptidyl mimic of superoxide dismutase with
therapeutic activity in rats. Science 1999;286:304–6.