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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

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

Received 9 June 2005; accepted 22 October 2005

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).

0954-6111/$ - see front matter & 2005 Elsevier Ltd. All rights reserved.
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-

cules play a role as secondary antioxidants. To the
Asthma is a chronic inflammatory airway disease.1 best of our knowledge, the levels of antioxidants in
The inflammatory cells infiltrating the airways the saliva of asthmatic children have not been
produce several mediators that modulate the previously studied.
inflammatory response. In recent years, oxidative The aim of this study was to analyze the
stress has been increasingly recognized as one of antioxidant status of saliva in children with asthma
the major factors contributing to the chronic during attack and remission, and to make a
inflammatory process. Furthermore, there is an comparison with the levels of salivary antioxidants
increase in generation of oxidants and lipid in healthy control children.
peroxidation products in the plasma, including a
range of toxic reactive oxygen species (ROS) such as
superoxide radical, hydrogen peroxide, hypochlor-
Patients & methods
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
Children presenting to the Pediatric Emergency
pathophysiological changes that are relevant in
Room (ER) of Meyer Children’s Hospital with acute
asthma, such as increased lipid peroxidation, in-
asthma were eligible to participate in the study.
creased airway reactivity and secretions, increased
Inclusion criteria were: age 6–18 years, asthma
production of chemoattractants, and increased
diagnosed according to GINA (Global Initiative for
vascular permeability.6–8 The elevated oxidative
Asthma) 2000 guidelines, and ability to perform
stress in asthma is also reflected by increased
spirometry consistently. Exclusion criteria were:
production of protein carbonyls in plasma,9 increased
acute gingivitis, acute febrile illness or clinical
plasma isoprostanes,10 increased oxidized glu-
pneumonia, any other chronic lung disease, and
tathione in bronchoalveolar lavage (BAL) fluid,11
administration of anti-inflammatory agents (in-
and increased production of NO in exhaled air.12
haled/ systemic corticosteroids or antileuko-
The lungs and blood contain several antioxidants
trienes) within 2 weeks or bronchodilator
to counter oxidant-mediated toxicity, including
administration less than 4 h prior to enrollment.
superoxide dismutase (SOD), catalase, glutathione,
Each subject was assessed during the acute
glutathione peroxidase (GSH-Px), catalase and
presentation to the ER, and 2–4 weeks later.
vitamins.13–16 Changes in antioxidant defenses have
Assessment consisted of a clinical score (CS),
been reported, including decreased GSH-Px in
spirometry, sialometry and sialochemistry.
whole blood, plasma, and platelets, a deficiency
of selenium,17–19 decreased protein sulfhydrils and
total antioxidant capacity in plasma,19 decreased Clinical assessment
SOD activity in BAL cells,20 and decreased vitamin C
and vitamin E concentration in BAL fluid.11 CS included respiratory rate, retractions, oxygen
Human saliva has a profound antioxidant capa- saturation, inspiratory/expiratory (I/E) ratio (esti-
city. The salivary antioxidant system includes mated by auscultation) and presence of inspiratory
various molecules and enzymes, of which the most or expiratory wheezing, each graded from 0 to 3,
important are the uric acid molecule and the maximum of 15 (Table 1).25 All measurements
peroxidase enzyme (salivary peroxidase (SPO)), during attacks and remissions were performed in
both of which are water-soluble. The lipid-soluble the Pediatric Pulmonology Unit, Rambam Medical
antioxidants carried by lipoproteins, whose con- Center, by the same investigator (LB) for consis-
centration in saliva is very low, contribute no more tency. Spirometry: forced expiratory volume in 1 s
than 10% of the total salivary antioxidant capa- (FEV1) was measured with a Vitalograph Alpha
city.21,22 Uric acid, the most important antioxidant spirometer (Vitalograph, Buckingham, UK). Healthy
molecule in saliva,22–24 contributes approximately children without any history or symptoms of asthma
70% of the total salivary antioxidant capacity, served as a control group.
Salivary oxidative stress in asthma 1197

Table 1 Clinical scores of asthmatic patients—each variable is graded between 0 and 3.

Score Wheezing Insp/Exp ratio Oxygen Accessory Respiratory rate

saturation muscles use

0 None 2:1 99–100  o20

1 End expiratory 1:1 96–98 + 20–35
2 Throughout expiration 1:2 93–95 ++ 36–50
3 Expiratory and inspiratory 1:3 o93 +++ 450

Salivary analysis assay, uric acid is transformed by uricase into

allantoin and hydrogen peroxide that, under the
Unstimulated whole saliva samples were obtained catalytic influence of peroxidase, oxidizes the
and no oral stimulus was permitted for 60 min prior chromogen (4-minophenazone/N-ethyl-methylani-
to collection, as previously described.24 Following lin propan-sulphonate sodic) to form a red com-
collection on ice, the salivary samples were frozen pound, whose intensity of color is proportional to
at 20 1C for subsequent analysis. Salivary gland the amount of uric acid present in the sample,
flow rates were expressed as volume of saliva (mL) which is read at a wavelength of 546 nm.
secreted per minute. The general sialochemistry
analysis was performed as previously described, Salivary total antioxidant status (TAS)
including: pH, calcium (Ca), phosphate (P), mag-
nesium (Mg), zinc (Zn), uric acid (UA), total protein The assay used was based on a commercial kit
(TP), albumin (Alb), lactate dehydrogenase (LDH) supplied by Randox (USA) in which metmyoglobin in
and amylase (Amy).24 The salivary analysis also the presence of iron is turned into ferrylmyoglobin.
included the following antioxidants: peroxidase, Incubation of the latter with the Randox reagent
SOD, uric acid and total antioxidant status (TAS) ABTS results in the formation of a blue-green
concentration.26,27 colored radical which can be detected at 600 nm.24
Salivary peroxidase (SPO) activity
Statistical analysis
SPO activity was measured using the thionitroben-
zoicacid (NBS) assay. Briefly, the calorimetric Data concerning the levels of various parameters
change induced by the reaction between the evaluated in whole saliva are expressed as means
enzyme and the substrate, Dithiobis 2-Nitrobensoic and ranges, and median7SEM. Paired t-test was used
Acid (DTNB) in the presence of mercaptoethanol, to compare salivary composition and antioxidant
was read at 412 nm wavelength for 20 s.24 levels between asthma attack and remission. Un-
paired t-test was used to compare between patients
Salivary superoxide dismutase (SOD) activity and control groups. Spearman’s rank coefficient of
correlation was used for analyzing saliva and the
Total activity of SOD isoenzymes (Cu0 -{1-[(phenyla- various clinical and spirometric parameters. Statis-
mino)-carbonyl]-3, 4-tetrazolium}-bis (4-methoxy- tical significance was set at Po0:05.
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 Results
superoxide anion (O 2 .) that is generated by
xanthine oxidase. The formazan is read at 470 nm. Twelve asthmatic children were recruited to the
SOD inhibits this reaction by scavenging the .O2. study, and were compared to 14 control patients.
One unit of the enzyme is defined as the amount of All asthmatic children presented to the ER with an
enzyme needed for 50% inhibition of absorption in acute exacerbation of asthma. Their ages ranged
the absence of the enzyme.27 from 6 to 18 years, with a mean7SD age of
9.7573.22 years. Nine patients had clinical man-
Salivary uric acid concentration ifestations of atopy: allergic rhinitis (9/9), atopic
dermatitis (4/9), and allergic conjunctivitis (6/9).
Uric acid concentration was measured with a kit Seven patients had a family history of asthma and
supplied by Sentinel CH (Milan, Italy).24 In this seven had passive smoking exposure.
1198 L. Bentur et al.

Table 2 Clinical scores and FEV1 during exacerbation and remission of asthma.

N ¼ 12 Mean7SD (Range)

Age (years) 9.7573.22 6–16

CS, attack 6.9274.58 1–14
CS, remission (N ¼ 10) 1.570.71 1–3
FEV1, attack (% predicted) 61719.01 34–87
FEV1, remission (% predicted) (N ¼ 10) 92.3722.33 66–139

CS—clinical score; FEV1—forced expiratory volume in 1 s.


All children received an oral tapering course of yet the uric acid concentration following remission
methylprednisolone (initial dose of 1–2 mg/kg) increased by 39% (Table 4). No correlation was
followed by maintenance therapy with budesonide found between saliva antioxidant levels and FEV1 or
Turbohaler (200 mcg twice daily). CS values.
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) Discussion
(Table 2).
The main finding of this study is that SPO was shown
to be reduced during acute asthmatic attack. This
Sialometry and sialochemistry
reduction may result from an increase in the level
of oxidants, which reacted with the enzyme.28,29
The mean salivary flow rate value and pH of the
This may be of paramount importance, since SPO is
entire group of patients was within normal values
considered the major salivary antioxidant enzyme.
and did not differ from the healthy controls. The
All other antioxidants measured exhibited a similar
sialochemical analysis demonstrated a significant
trend, with increased levels during remission,
decrease of calcium and increase of phosphate
although not of statistical significance.
concentrations in the asthmatic children (Tables 3
The mechanism by which the decreased levels of
and 4). The amylase activity was twofold higher in
calcium concentration and increased phosphate
asthmatic children during attack (P ¼ 0:09). No
levels occurred in asthmatic children remains
significant differences were found between pa-
obscure and should be further elucidated. The
tients and controls regarding total protein, albumin
decreased levels of SPO and calcium, and increased
levels, and LDH activity (Tables 3 and 4).
level of phosphate observed are in accord with the
studies published by Ryberg et al.30 who reported
Salivary antioxidant analysis changes in the salivary composition of asthmatic
patients treated with beta 2-adrenoceptor ago-
The salivary antioxidant analysis revealed a sig- nists. This significant decrease in the SPO leaves
nificant decrease in the levels of SPO in asthmatic the oral cavity exposed to oxidative stress. More-
patients during acute asthma attack compared with over, Hyyppa et al.31 reported that asthmatic
controls (Fig. 1). The levels of SPO increased during children had more gingivitis than their healthy
remission of attack without reaching control levels, controls. Previous studies32–34 suggested an asso-
although this difference was statistically insignif- ciation between salivary antioxidant activity and
icant. A similar trend toward increasing levels while periodontal disease.
in remission was observed for all other antioxidants Asthma, as well as other pulmonary diseases,
(TAS, uric acid and SOD). However, it also did not have long been associated with inflammation, and
achieve statistical significance. Thus, the SOD recently with oxidative stress. Oxidative stress is
activity values and TAS levels dropped by 26% and the final result of numerous molecular pathways
28%, respectively, in asthmatic patients during involving ROS and RNS, which may lead to the
attack, as compared to healthy controls. Following observed increased airway reactivity and secretions,
remission, these values fully recovered (Table 3). increased production of chemoattractants, and
No significant differences were found between increased vascular permeability.6–8 Although the
patients and controls regarding salivary uric acid, aforementioned oxidative process mainly affects
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).


the respiratory system, changes in the oxidative– Reviewing antioxidant function in the oxidative
antioxidative balance can also be seen in the plasma process shows that it has conflicting functions: on
and blood cells.3,9,10. the one hand, it participates in the scavenging of
With the profound antioxidative capacity of the potent superoxide radical, but on the other
human saliva in mind, our study aimed to evaluate hand, it creates the hydrogen peroxide radical
salivary oxidative status in asthmatic children. The which propagates further ROS and RNS creation.
current study demonstrates that asthmatic children The net result of these conflicting functions is
have decreased salivary antioxidant levels during naturally determined by other factors, which are
acute asthma exacerbations, compared to healthy not well understood and need further research for
children, as reflected by decreased salivary SPO clarification.
levels. Similar to our finding, Smith et al.20 found We demonstrated that SPO levels increased
decreased antioxidant level (SOD) activity in BAL toward normal levels by 2–4 weeks of ambulatory
cells of asthmatic patients, while two recently anti-inflammatory therapy, in association with
published works found increased SOD activity in significant clinical and laboratory-confirmed im-
asthmatic patients’ erythrocytes.28,29 The differ- provement. Corticosteroids used in asthma treat-
ence in the findings may be explained by the ment suppress airway inflammation. It is possible
difference in the tissues obtained. Recently, Schock that anti-inflammatory therapy alleviates the oxi-
et al.35 demonstrated significantly lower levels of dative load by inhibiting airway inflammation, and
ascorbic acid in induced sputum and saliva com- thereby suppresses the salivary oxidative process.
pared with plasma, and suggested that there is no This study has several limitations. The study
free diffusion of ascorbic acid between the vascu- population is relatively small. The antioxidative
lar-interstitial fluid compartments and the tracheo- parameters were measured in the saliva without
bronchial airway secretions. comparable antioxidative parameters in other body
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
salivary antioxidants in children with asthma.
200 Asthma Attack
Asthma Remission
*p<0.05 Healthy
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