ARTICLE IN PRESS

Respiratory Medicine (2006) 100, 1195–1201

Salivary oxidative stress in children during acute

asthmatic attack and during remission
Lea Bentura,b,, Yasser Mansoura, Riva Brikb,c,
Yoav Eizenberga, Rafael M. Naglerb,d

a
Pediatric Pulmonology Unit, Meyer Children’s Hospital, Rambam Medical Center,
P.O. Box 9602, Haifa 31092, Israel
b
Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel
c
Pediatric Department, Meyer Children’s Hospital, Rambam Medical Center
d
Department of Oral and Maxillofacial Surgery, Oral Biochemistry Laboratory and Salivary Clinic,
Rambam Medical Center

Received 9 June 2005; accepted 22 October 2005

KEYWORDS Summary
Asthma; Background: Patients with asthma generate an increased amount of reactive oxygen
Saliva; species from peripheral blood cells, which may contribute to its pathogenesis. Saliva
Oxidative stress analysis is non-invasive and friendly to children. We undertook this study to analyze
the salivary oxidative profile and composition in children with asthma during attack
and remission, and to compare them with the levels of salivary antioxidants of
healthy control children.
Methods: School age (range 6–18 years) children referred to the emergency room
for acute asthma were included. Clinical score was assessed, spirometry performed,
and saliva samples were collected and analyzed. All measurements were repeated
during remission of asthma attack (2–4 weeks after attack). Salivary analysis was
performed blindly during asthma attack and the results were compared to those
obtained during remission, and to those of the control group.
Results: Statistically significant decreases in levels of salivary peroxidase enzyme
activity were observed in asthmatic children during attack compared with healthy
controls, with partial recovery during remission of attack. Similarly decreased levels
of calcium concentrations were observed in asthmatic children, accompanied by
increased phosphate levels.
Conclusions: Children with acute asthma attacks exhibit a decrease in the activity
of the most important salivary antioxidant enzyme-peroxidase, which is accom-
panied by other salivary composition alterations. Hence, acute asthma is manifested

Corresponding author. Pediatric Pulmonology Unit, Meyer Children’s Hospital, Rambam Medical Center, P.O. Box 9602, Haifa 31092,
Israel. Tel.: +972 4 8543263; fax: +972 4 8543127.
E-mail address: l_bentur@rambam.health.gov.il (L. Bentur).

0954-6111/$ - see front matter & 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.rmed.2005.10.022
 ARTICLE IN PRESS
1196 L. Bentur et al.

by salivary changes. This implies systemic oxidative stress in asthma, which may be
reflected in salivary analysis.
& 2005 Elsevier Ltd. All rights reserved.

Introduction according to Moore,22 while ascorbic acid mole-

Asthma is a chronic inflammatory airway disease.1
The inflammatory cells infiltrating the airways
produce several mediators that modulate the
inflammatory response. In recent years, oxidative
stress has been increasingly recognized as one of
the major factors contributing to the chronic
inflammatory process. Furthermore, there is an
increase in generation of oxidants and lipid
peroxidation products in the plasma, including a
range of toxic reactive oxygen species (ROS) such as
superoxide radical, hydrogen peroxide, hypochlor-
ous acid and hydroxyl radical.2–5 Moreover, these
ROS can react with nitrite and nitric oxide (NO) to
form reactive nitrogen species (RNS).
ROS have been shown to be associated with many
pathophysiological changes that are relevant in
asthma, such as increased lipid peroxidation, in-
creased airway reactivity and secretions, increased
production of chemoattractants, and increased
vascular permeability.6–8 The elevated oxidative
stress in asthma is also reflected by increased
production of protein carbonyls in plasma,9 increased
plasma isoprostanes,10 increased oxidized glu-
tathione in bronchoalveolar lavage (BAL) fluid,11
and increased production of NO in exhaled air.12
The lungs and blood contain several antioxidants
to counter oxidant-mediated toxicity, including
superoxide dismutase (SOD), catalase, glutathione,
glutathione peroxidase (GSH-Px), catalase and
vitamins.13–16 Changes in antioxidant defenses have
been reported, including decreased GSH-Px in
whole blood, plasma, and platelets, a deficiency
of selenium,17–19 decreased protein sulfhydrils and
total antioxidant capacity in plasma,19 decreased
SOD activity in BAL cells,20 and decreased vitamin C
and vitamin E concentration in BAL fluid.11
Human saliva has a profound antioxidant capa-
city. The salivary antioxidant system includes
various molecules and enzymes, of which the most
important are the uric acid molecule and the
peroxidase enzyme (salivary peroxidase (SPO)),
both of which are water-soluble. The lipid-soluble
antioxidants carried by lipoproteins, whose con-
centration in saliva is very low, contribute no more
than 10% of the total salivary antioxidant capa-
city.21,22 Uric acid, the most important antioxidant
molecule in saliva,22–24 contributes approximately
70% of the total salivary antioxidant capacity,
cules play a role as secondary antioxidants. To the
best of our knowledge, the levels of antioxidants in
the saliva of asthmatic children have not been
previously studied.
The aim of this study was to analyze the
antioxidant status of saliva in children with asthma
during attack and remission, and to make a
comparison with the levels of salivary antioxidants
in healthy control children.
Patients & methods
Patients
Children presenting to the Pediatric Emergency
Room (ER) of Meyer Children’s Hospital with acute
asthma were eligible to participate in the study.
Inclusion criteria were: age 6–18 years, asthma
diagnosed according to GINA (Global Initiative for
Asthma) 2000 guidelines, and ability to perform
spirometry consistently. Exclusion criteria were:
acute gingivitis, acute febrile illness or clinical
pneumonia, any other chronic lung disease, and
administration of anti-inflammatory agents (in-
haled/ systemic corticosteroids or antileuko-
trienes) within 2 weeks or bronchodilator
administration less than 4 h prior to enrollment.
Each subject was assessed during the acute
presentation to the ER, and 2–4 weeks later.
Assessment consisted of a clinical score (CS),
spirometry, sialometry and sialochemistry.
Clinical assessment
CS included respiratory rate, retractions, oxygen
saturation, inspiratory/expiratory (I/E) ratio (esti-
mated by auscultation) and presence of inspiratory
or expiratory wheezing, each graded from 0 to 3,
maximum of 15 (Table 1).25 All measurements
during attacks and remissions were performed in
the Pediatric Pulmonology Unit, Rambam Medical
Center, by the same investigator (LB) for consis-
tency. Spirometry: forced expiratory volume in 1 s
(FEV1) was measured with a Vitalograph Alpha
spirometer (Vitalograph, Buckingham, UK). Healthy
children without any history or symptoms of asthma
served as a control group.
 ARTICLE IN PRESS
Salivary oxidative stress in asthma
Table 1 Clinical scores of asthmatic patients—each variable is graded between 0 and 3.
Score Wheezing Insp/Exp ratio
0 None 2:1
1 End expiratory 1:1
2 Throughout expiration 1:2
3 Expiratory and inspiratory 1:3
Salivary analysis
Unstimulated whole saliva samples were obtained
and no oral stimulus was permitted for 60 min prior
to collection, as previously described.24 Following
collection on ice, the salivary samples were frozen
at
20 1C for subsequent analysis. Salivary gland
flow rates were expressed as volume of saliva (mL)
secreted per minute. The general sialochemistry
analysis was performed as previously described,
including: pH, calcium (Ca), phosphate (P), mag-
nesium (Mg), zinc (Zn), uric acid (UA), total protein
(TP), albumin (Alb), lactate dehydrogenase (LDH)
and amylase (Amy).24 The salivary analysis also
included the following antioxidants: peroxidase,
SOD, uric acid and total antioxidant status (TAS)
concentration.26,27
Salivary peroxidase (SPO) activity
SPO activity was measured using the thionitroben-
zoicacid (NBS) assay. Briefly, the calorimetric
change induced by the reaction between the
enzyme and the substrate, Dithiobis 2-Nitrobensoic
Acid (DTNB) in the presence of mercaptoethanol,
was read at 412 nm wavelength for 20 s.24
Salivary superoxide dismutase (SOD) activity
Total activity of SOD isoenzymes (Cu0 -{1-[(phenyla-
mino)-carbonyl]-3, 4-tetrazolium}-bis (4-methoxy-
6-nitro) benzenesulfonic acid hydrate reduction by
xanthine–xanthine oxidase. The method is a mod-
ification of the NBT assay. Xtt is reduced by the
superoxide anion (O
2 .) that is generated by
xanthine oxidase. The formazan is read at 470 nm.
SOD inhibits this reaction by scavenging the .O2.
One unit of the enzyme is defined as the amount of
enzyme needed for 50% inhibition of absorption in
the absence of the enzyme.27
Salivary uric acid concentration
Uric acid concentration was measured with a kit
supplied by Sentinel CH (Milan, Italy).24 In this
1197
Oxygen Accessory Respiratory rate
saturation muscles use
99–100
o20
96–98 + 20–35
93–95 ++ 36–50
o93 +++ 450
assay, uric acid is transformed by uricase into
allantoin and hydrogen peroxide that, under the
catalytic influence of peroxidase, oxidizes the
chromogen (4-minophenazone/N-ethyl-methylani-
lin propan-sulphonate sodic) to form a red com-
pound, whose intensity of color is proportional to
the amount of uric acid present in the sample,
which is read at a wavelength of 546 nm.
Salivary total antioxidant status (TAS)
The assay used was based on a commercial kit
supplied by Randox (USA) in which metmyoglobin in
the presence of iron is turned into ferrylmyoglobin.
Incubation of the latter with the Randox reagent
ABTS results in the formation of a blue-green
colored radical which can be detected at 600 nm.24
Statistical analysis
Data concerning the levels of various parameters
evaluated in whole saliva are expressed as means
and ranges, and median7SEM. Paired t-test was used
to compare salivary composition and antioxidant
levels between asthma attack and remission. Un-
paired t-test was used to compare between patients
and control groups. Spearman’s rank coefficient of
correlation was used for analyzing saliva and the
various clinical and spirometric parameters. Statis-
tical significance was set at Po0:05.
Results
Twelve asthmatic children were recruited to the
study, and were compared to 14 control patients.
All asthmatic children presented to the ER with an
acute exacerbation of asthma. Their ages ranged
from 6 to 18 years, with a mean7SD age of
9.7573.22 years. Nine patients had clinical man-
ifestations of atopy: allergic rhinitis (9/9), atopic
dermatitis (4/9), and allergic conjunctivitis (6/9).
Seven patients had a family history of asthma and
seven had passive smoking exposure.
 ARTICLE IN PRESS
1198
Table 2 Clinical scores and FEV1 during exacerbation and remission of asthma.
N ¼ 12
Age (years)
CS, attack
CS, remission (N ¼ 10)
FEV1, attack (% predicted)
FEV1, remission (% predicted) (N ¼ 10)
CS—clinical score; FEV1—forced expiratory volume in 1 s.
 Po0:05.
All children received an oral tapering course of
methylprednisolone (initial dose of 1–2 mg/kg)
followed by maintenance therapy with budesonide
Turbohaler (200 mcg twice daily).
CSs decreased from 6.9274.58 during attack to
1.570.71 (mean7SD) on remission (Po0:0001),
while FEV1 rose from 61719.01 to 92.3722.33
percent of predicted, respectively (Po0:0001)
(Table 2).
Sialometry and sialochemistry
The mean salivary flow rate value and pH of the
entire group of patients was within normal values
and did not differ from the healthy controls. The
sialochemical analysis demonstrated a significant
decrease of calcium and increase of phosphate
concentrations in the asthmatic children (Tables 3
and 4). The amylase activity was twofold higher in
asthmatic children during attack (P ¼ 0:09). No
significant differences were found between pa-
tients and controls regarding total protein, albumin
levels, and LDH activity (Tables 3 and 4).
Salivary antioxidant analysis
The salivary antioxidant analysis revealed a sig-
nificant decrease in the levels of SPO in asthmatic
patients during acute asthma attack compared with
controls (Fig. 1). The levels of SPO increased during
remission of attack without reaching control levels,
although this difference was statistically insignif-
icant. A similar trend toward increasing levels while
in remission was observed for all other antioxidants
(TAS, uric acid and SOD). However, it also did not
achieve statistical significance. Thus, the SOD
activity values and TAS levels dropped by 26% and
28%, respectively, in asthmatic patients during
attack, as compared to healthy controls. Following
remission, these values fully recovered (Table 3).
No significant differences were found between
patients and controls regarding salivary uric acid,
L. Bentur et al.
Mean7SD (Range)
9.7573.22 6–16
6.9274.58 1–14
1.570.71 1–3
61719.01 34–87
92.3722.33 66–139
yet the uric acid concentration following remission
increased by 39% (Table 4). No correlation was
found between saliva antioxidant levels and FEV1 or
CS values.
Discussion
The main finding of this study is that SPO was shown
to be reduced during acute asthmatic attack. This
reduction may result from an increase in the level
of oxidants, which reacted with the enzyme.28,29
This may be of paramount importance, since SPO is
considered the major salivary antioxidant enzyme.
All other antioxidants measured exhibited a similar
trend, with increased levels during remission,
although not of statistical significance.
The mechanism by which the decreased levels of
calcium concentration and increased phosphate
levels occurred in asthmatic children remains
obscure and should be further elucidated. The
decreased levels of SPO and calcium, and increased
level of phosphate observed are in accord with the
studies published by Ryberg et al.30 who reported
changes in the salivary composition of asthmatic
patients treated with beta 2-adrenoceptor ago-
nists. This significant decrease in the SPO leaves
the oral cavity exposed to oxidative stress. More-
over, Hyyppa et al.31 reported that asthmatic
children had more gingivitis than their healthy
controls. Previous studies32–34 suggested an asso-
ciation between salivary antioxidant activity and
periodontal disease.
Asthma, as well as other pulmonary diseases,
have long been associated with inflammation, and
recently with oxidative stress. Oxidative stress is
the final result of numerous molecular pathways
involving ROS and RNS, which may lead to the
observed increased airway reactivity and secretions,
increased production of chemoattractants, and
increased vascular permeability.6–8 Although the
aforementioned oxidative process mainly affects
 ARTICLE IN PRESS
Salivary oxidative stress in asthma 1199

Table 3 Comparison of salivary pH, electrolytes, protein, enzyme levels and antioxidant activity levels
between healthy controls and asthmatic patients during remission.

Healthy (n ¼ 14) Asthma in remission (n ¼ 12)

pH Median (range) 7.00 (6.4–7.3) 7.00 (6.4–7.0)

Mean7SEM 6.9470.05 6.8770.07
Ca (mg/dL) Median (range) 2.35(0.5–59.7) 0.70 (0.2–1.2)
Mean7SEM 6.6974.10 0.7470.11
P (mg/dL) Median (range) 11.4 (4.9–1.5) 14.2 (9.5–20.2)
Mean7SEM 14.2473.00 14.4071.07
Mg (mg/dL) Median (range) 0.95 (0.4–1.9) 0.7 (0.2–1.2)
Mean7SEM 1.0070.13 0.7270.11
Zn (mg/dL) Median (range) 132 (21–586) 145 (7.0–314)
Mean7SEM 165741.60 144734.50
TP (mg/dL) Median (range) 67.05 (16.6–100.8) 71.1(44.4–126.6)
Mean7SEM 62.2076.23 7477.90
ALB (mg/dL) Median (range) 39.4 (10.1–247.4) 51.3 (7.2–83.9)
Mean7SEM 61.65716.25 47.9077.87
LDH(U/L) Median (range) 422 (42–912) 509 (108–831)
Mean7SEM 424778 509776.00
AMY (IU/L) Median (range) 663 (110–1192) 830 (179.3–2802)
Mean7SEM 716780.8 9907406.
Uric acid (mg/dL) Median (range) 1.83 (0.32–20.38) 2.47 (0.62–3.66)
Mean7SEM 3.3871.35 2.1870.36
SPO (U/100 mL) Median (range) 580 (420–890) 540 (430–670)
Mean7SEM 600740 540748
TAS (mmol/L) Median (range) 0.5 (0.13–1.06) 0.61 (0.24–0.95)
Mean7SEM 0.570.07 0.6070.09
SOD (U/mL) Median (range) 1.71 (0.32–7.34) 1.71 (0.34–4.66)
Mean7SEM 2.1570.55 1.9270.62

Results are expressed as medians and range, means7SEM (standard error).

 Po0:05.

the respiratory system, changes in the oxidative–
antioxidative balance can also be seen in the plasma
and blood cells.3,9,10.
With the profound antioxidative capacity of
human saliva in mind, our study aimed to evaluate
salivary oxidative status in asthmatic children. The
current study demonstrates that asthmatic children
have decreased salivary antioxidant levels during
acute asthma exacerbations, compared to healthy
children, as reflected by decreased salivary SPO
levels. Similar to our finding, Smith et al.20 found
decreased antioxidant level (SOD) activity in BAL
cells of asthmatic patients, while two recently
published works found increased SOD activity in
asthmatic patients’ erythrocytes.28,29 The differ-
ence in the findings may be explained by the
difference in the tissues obtained. Recently, Schock
et al.35 demonstrated significantly lower levels of
ascorbic acid in induced sputum and saliva com-
pared with plasma, and suggested that there is no
free diffusion of ascorbic acid between the vascu-
lar-interstitial fluid compartments and the tracheo-
bronchial airway secretions.
Reviewing antioxidant function in the oxidative
process shows that it has conflicting functions: on
the one hand, it participates in the scavenging of
the potent superoxide radical, but on the other
hand, it creates the hydrogen peroxide radical
which propagates further ROS and RNS creation.
The net result of these conflicting functions is
naturally determined by other factors, which are
not well understood and need further research for
clarification.
We demonstrated that SPO levels increased
toward normal levels by 2–4 weeks of ambulatory
anti-inflammatory therapy, in association with
significant clinical and laboratory-confirmed im-
provement. Corticosteroids used in asthma treat-
ment suppress airway inflammation. It is possible
that anti-inflammatory therapy alleviates the oxi-
dative load by inhibiting airway inflammation, and
thereby suppresses the salivary oxidative process.
This study has several limitations. The study
population is relatively small. The antioxidative
parameters were measured in the saliva without
comparable antioxidative parameters in other body
 ARTICLE IN PRESS
1200 L. Bentur et al.

Table 4 Comparison of salivary pH, electrolytes, protein, enzyme levels and antioxidant activity between
asthmatic patients during attack and remission.

Asthma attack (n ¼ 12) Asthma in remission (n ¼ 12)

pH Median (range) 7.00 (6.7–7.6) 7.00 (6.4–7.0)

Mean7SEM 7.0070.07 6.8770.07
Ca (mg/dL) Median (range) 0.70 (0.3–39.2) 0.70 (0.2–1.2)
Mean7SEM 4.3473.18 0.7470.11
P (mg/dL) Median (range) 13.85 (7.5–22.8) 14.2 (9.5–20.2)
Mean7SEM 14.3271.10 14.4071.07
Mg (mg/dL) Median (range) 0.65 (0.2–2.8) 0.7 (0.2–1.2)
Mean7SEM 0.8270.21 0.7270.11
Zn (mg/dL) Median (range) 134 (12–331) 145 (7.0–314)
Mean7SEM 165741.60 144734.50
TP (mg/dL) Median (range) 66.35 (9.7–243.3) 71.1(44.4–126.6)
Mean7SEM 62.2076.23 7477.90
ALB (mg/dL) Median (range) 43.55 (7.6–78.8) 51.3 (7.2–83.9)
Mean7SEM 62.07721.6 47.9077.87
LDH (U/L) Median (range) 686 (31–983) 509 (108–831)
Mean7SEM 585797 509776.00
AMY (IU/L) Median (range) 1234 (235–2674) 830 (179.3–2802)
Mean7SEM 14367957 9907406.
Uric acid (mg/dL) Median(range) 1.78 (0.69–2.68) 2.47 (0.62–3.66)
Mean7SEM 1.7370.16 2.1870.36
SPO (U/100 mL) Median (range) 500 (280–680) 540 (430–670)
Mean7SEM 480730 540748
TAS (mmol/L) Median (range) 0.36 (0.22–1.85) 0.61 (0.24–0.95)
Mean7SEM 0.670.15 0.6070.09
SOD (U/mL) Median (range) 1.27 (0.31–7.09) 1.71(0.34–4.66)
Mean7SEM 2.1570.65 1.9270.62

Results are expressed as medians and range, means7SEM (standard error).

1000 convincing evidence that free radical mechanisms

480±30* 540±48 600±40*
are involved in the pathogenesis of asthma and that
800 antioxidants may be protective.
Further studies involving larger groups comparing
SPO (U/100µL)

600 saliva, serum and BAL parameters should be

conducted in order to fully understand the role of
400
salivary antioxidants in children with asthma.
200 Asthma Attack
Asthma Remission
*p<0.05 Healthy
0
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